Local and Systemic Proteomic Changes in - ACS Publications

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Local and Systemic Proteomic Changes in Medicago Truncatula at an Early Phase of Sinorhizobium meliloti Infection Barbara Molesini,† Daniela Cecconi,† Youry Pii, and Tiziana Pandolfini* Department of Biotechnology, University of Verona, Strada Le Grazie, 15, Verona 37134, Italy S Supporting Information *

ABSTRACT: A symbiotic association with N-fixing bacteria facilitates the growth of leguminous plants under nitrogenlimiting conditions. The establishment of the symbiosis requires signal exchange between the host and the bacterium, which leads to the formation of root nodules, inside which bacteria are hosted. The formation of nodules is controlled through local and systemic mechanisms, which involves rootshoot communication. Our study was aimed at investigating the proteomic changes occurring in shoots and concomitantly in roots of Medicago truncatula at an early stage of Sinorhizobium meliloti infection. The principal systemic effects consisted in alteration of chloroplast proteins, induction of proteins responsive to biotic stress, and changes in proteins involved in hormonal signaling and metabolism. The most relevant local effect was the induction of proteins involved in the utilization of photosynthates and C-consuming processes (such as sucrose synthase and fructose-bisphosphate aldolase). In addition, some redox enzymes such as peroxiredoxin and ascorbate peroxidase showed an altered abundance. The analysis of local and systemic proteome changes suggests the occurrence of a stress response in the shoots and the precocious alteration of energy metabolism in roots and shoots. Furthermore, our data indicate the possibility that ABA and ethylene participate in the communicative network between root and shoot in the control of rhizobial infection. KEYWORDS: rhizobial symbiosis, systemic response, proteome, root-shoot communication

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INTRODUCTION Nitrogen is an essential element for plant growth, development, and reproduction. In nitrogen-deprived soils, leguminous plants may associate with compatible microorganisms, referred to as rhizobia. The mutualistic relationship is manifested by the production of root nodules, inside which rhizobia convert atmospheric nitrogen into ammonia. The symbiosis is beneficial for both organisms: bacteria offer exploitable nitrogen compound to plants, and plants in turn supply bacteria with carbon sources. This unique capacity allows legumes to propagate under nitrogen limiting conditions. The environmental and agricultural benefits of legumes have been recognized for centuries, but the increasing public demand for food leads to the widespread adoption of synthetic nitrogen fertilizers. Their inefficient application has negative environmental and economic impacts.1,2 Besides this, their price has increased because the energy consumed in their production derives from non-renewable sources.1,2 Biological nitrogen fixation is environmentally friendly because it depends on renewable energy sources. Therefore, legumes’ use in sustainable agriculture should be maximized as a natural source of nitrogen for crops. Significant progress has been made to get deeper insight into the physiological and molecular aspects of root interaction with symbiotic organisms for both heuristic and applied interest. Better knowledge about © 2013 American Chemical Society

this process could allow improving legume symbiotic performance and transferring legume benefits into non-nodulating crops. The control of host−symbiont interaction and nodule development is complex and requires a continuous exchange of signals between the partners. Rhizobial infection takes place in the epidermis at the root hair level.3 Plants release flavonoids into the soil that stimulate the bacterial synthesis of chitin derivatives called nodulation factors (NFs). NFs elicit rootspecific pathways that are crucial to regulate organogenesis as well as the number of nodules. In the symbiosis between Medicago truncatula and Sinorhizobium meliloti, the LysMReceptor Like Kinases (LysM-RLK) LYK3 and NFP mediate the recognition of bacterial NFs.4−6 After NFs perception, a downstream signal transduction cascade, termed the common signaling pathway, is induced in both epidermis and subtended cortical cells.4−7 In the root hair cells, perception of NFs determines a rapid influx of Ca2+ and a spiking in cytosolic Ca2+ concentration in the nucleus and perinuclear region.8−10 NFs perception results also in a physical deformation of the root hair tips that curl to entrap the bacterial colony.11,12 Generation of the calcium spiking requires ion channel proteins and Received: May 20, 2013 Published: December 18, 2013 408

dx.doi.org/10.1021/pr4009942 | J. Proteome Res. 2014, 13, 408−421

Journal of Proteome Research

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through transcriptomic and proteomic analyses of the xylem sap.45−47 In the present work, M. truncatula and S. meliloti symbiosis is investigated at an early stage of infection. We compared the proteome of M. truncatula root apparatuses inoculated with rhizobia with that of mock-inoculated roots. Considering that we are examining the entire roots, this comparative analysis would be helpful to find the most prominent metabolic changes underlying the early phase of the symbiosis. In addition, we conducted a comparative proteomic analysis on M. truncatula shoots upon rhizobial infection to identify systemic changes in proteins’ abundance. The simultaneous analysis of shoot and root proteome of rhizobia-infected M. truncatula represents a new approach in the study of metabolic coordination of the two organs during the symbiotic interaction.

leucine-rich repeat receptor-like kinase, while the calcium spiking signal perception involves calcium calmodulin-dependent protein kinases (CCaMK).7 After binding of Ca2+ and Ca2+/calmodulin, CCaMK induces the epidermal expression of specific early nodulin genes via the phosphorylation of transcription factors. Concomitantly to epidermal responses, the cortical cells are activated leading to the formation of nodule primordia.13 In curled root hairs, tubular infection threads are formed through which the bacteria enter the plant. Infection threads progress and ramify toward the growing primordia in the cortex.14 Once released from the infection threads, rhizobia invade the primordia.15 Epidermal and cortical events are spatially separated but occur simultaneously and must be coordinated for a successful nodulation.16−19 The phytohormone cytokinin plays an important role in coordinating epidermal and cortical events.17,18 The plant controls the nodulation through a network of short- and long-distance signals that spread between root and shoot and vice versa. Nodule formation and nitrogen fixation are energetically expensive for the legume (i.e., 12−17 g of carbon per gram of nitrogen obtained), and if excessive, nodulation would restrict plant growth.20−22 Therefore, above a critical threshold level, existing functional nodules hamper the later establishment of successful infection events through a negative feedback regulation called autoregulation of nodulation (AON).9,23,24 The precise beginning of AON is unknown; however, several experimental data suggest AON is activated when the first nodules develop.25 Grafting experiments using supernodulating mutants defective in AON and wild-type plants demonstrate that the root phenotype is controlled by the shoot.22,26−28 In fact, a mutant shoot grafted onto a wild-type rootstock confers a mutant phenotype in the root, while the reciprocal graft allows normal regulation of the nodule number.22,29 This evidence suggests AON involves a systemic signaling pathway and shoot-controlled factors. The AON signal transferred from nodulating root to the shoot is perceived by a leucine-rich repeat receptor-like kinase (LRRRLK).20,27,30 Afterward, a second shoot-derived signal moves toward the root inhibiting later nodulation events.9,29−31 The LRR-RLKs display homology with CLAVATA 1 (CLV1) receptor kinase of Arabidopsis thaliana, which controls shoot and floral meristem size.32 The signals generated in the root in response to rhizobia inoculation, which systemically travel into the shoot activating CLV1-like receptor kinase, are CLAVATA3/endosperm-surrounding region (CLE) peptides.33 Besides these, other genes with yet unknown function are involved in AON response at the root level.21,34,35 To date, AON process is still only partially understood. Recent rapid progress has been made in plant biology, in particular with the introduction of high-throughput “omics” technologies. The success of these approaches applied to the symbiosis between M. truncatula and S. meliloti is linked to the availability of relatively complete genome sequences of both S. meliloti and of M. truncatula.36,37 Several comparative transcriptomic and proteomic analyses have been employed to investigate the molecular mechanisms of N-fixing symbiosis, mainly focusing on root, nodules, and portions of the roots corresponding to the infected zone.38−44 So far, none of the studies has considered the systemic proteomic changes occurring in M. truncatula shoots in response to S. meliloti infection. Differently, signal transmission from Glycine max nodulated root to aerial organs has been partially unraveled

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MATERIALS AND METHODS

Plant Material and Sinorhizobium meliloti Inoculation

Medicago truncatula seeds of cv Jemalong A17 were scarified, sterilized in 5% commercial bleach for 3 min, rinsed 3 times with sterile water, and in vitro germinated on 0.8% agar plates. The plates were maintained in darkness at 4 °C for 2 days and then placed in a growth chamber at a constant temperature of 25 °C with a 14 h/10 h light/dark cycle, with an average irradiance of 120 μmol m−2 s−1 of photosynthetically active radiation (PAR). Ten-day-old seedlings were transferred to small pots and grown on a sand and perlite mixture (1:1) in a growth chamber under a 14 h light/10 h dark regimen at 22/18 °C with an average irradiance of 120 μmol m−2 s−1 of PAR; the relative humidity was 65%. The plantlets were supplemented with a nitrogen-free nutrient solution (0.13 mM KH2PO4; 0.3 mM CaCl2·2H2O; 0.06 mM MgSO4·7H2O; 0.2 mM K2SO4; 0.014 mM FeNa EDTA; 1.56 μM H3BO3; 1.24 μM MnSO4· H2O; 4.5 μM KCl; 0.11 μM ZnSO4·7H2O; 0.1 μM CuSO4· 5H2O; 0.32 μM H2SO4; 2.1 μM Na2MoO4·2H2O), and sterile deionized water was added when necessary. Plant inoculation was performed with Sinorhizobium meliloti strain 1021, a streptomycin-resistant derivative of wild-type field isolate SU47.48 S. meliloti was grown overnight at 28 °C in liquid LBMC medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 2.6 mM MgSO4, 2.6 mM CaCl2) supplemented with 200 μg/mL streptomycin, then collected by centrifugation, and suspended in 10 mM MgSO4. The bacterial suspension was diluted with liquid BNM medium 49 to OD600 = 0.1 (corresponding to approximately 108 CFU mL−1). For plant inoculation, each seedling was placed in a single 50 mL tube containing the nitrogen-free nutrient solution plus perlite50 and treated with BNM medium-diluted bacteria. For control (mock-inoculated), the seedlings were treated with sterile BNM medium containing 10 mM MgSO4. Different sets of plants were grown and used for the various analyses. For microscopic analysis, plants were collected 18 h, 72 h, and 7 days after rhizobial inoculation. For proteomic analysis, plants were harvested 72 h after rhizobial inoculation; quantitative RT-PCR was performed at the same time point, unless otherwise specified. The roots were separated from the shoots, quickly frozen in liquid nitrogen, and stored at −80 °C. Microscopic Characterization of S. meliloti Infection

For the phenotypic characterization of the infection process, M. truncatula plants were inoculated with either S. meliloti 1021 wild-type or S. meliloti 1021 bearing pXLGD4, a plasmid containing the constitutive hemA::lacZ gene of Escherichia 409



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coli.51 The bacterial infection was visualized by histochemical staining of β-galactosidase activity, following a protocol previously described.52 Whole root samples were mounted on glass slides with coverslips and observed with a Leica DM2500 microscope equipped with a DFC420C digital camera (Leica Microsystems).

presence of Tris/glycine/SDS running buffer. The electrophoresis was conducted setting a current of 40 mA for each gel for 3 min, then 2 mA/gel for 1 h, and 20 mA/gel until bromophenol blue reached the bottom of the gel. Gels were fixed in 40% ethanol and 10% acetic acid for 30 min and then stained overnight at the room temperature on an orbital shaker with ultrasensitive Sypro Ruby solution (Bio-Rad). Gels were destained with 10% methanol and 7% acetic acid for 1 h and then rinsed with pure distilled water for 3 h.

Protein Extraction

Total protein extractions from shoots and roots were performed following the procedures described in http://www. noble.org/medicagohandbook/ and by Bestel-Corre et al.,39 respectively. For each treatment (mock-inoculation and rhizobial-inoculation), five protein samples were obtained from different pools of plant material derived from 20 individually inoculated plants. For total protein extraction from green tissues, samples were ground under liquid nitrogen in the presence of protease inhibitor cocktail (Sigma-Aldrich) with cold (4 °C) 10% TCA in acetone and 0.07% β-mercaptoethanol (1:10 w/v), incubated for at least 45 min at −20 °C for protein precipitation, and then centrifuged at 14,000g for 15 min at 4 °C. Protein pellets were washed 3 times in 90% acetone containing 0.07% βmercaptoethanol at 4 °C to remove any residual TCA traces that could have negative effects on isoelectrofocusing protein separation. After the last wash, pellets were air-dried and then dissolved in solubilization buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% Triton X-100, 20 mM Tris pH 8.0). For total protein extraction from roots, samples were ground under liquid nitrogen in the presence of protease inhibitor cocktail (Sigma-Aldrich) with a lysis buffer containing 0.7 M sucrose, 50 mM EDTA, 0.1 M KCl, 10 mM thiourea, 50 mM EDTA, 2% v/v β-mercaptoethanol, and 0.5 M Tris-HCl, pH 7.5. An equal volume of phenol saturated with 10 mM Tris buffer pH 8.0 and 1 mM EDTA was added. Samples were mixed for 30 min and then centrifuged at 12,000g for 30 min at 4 °C to separate and afterward recover the phenolic phase. Proteins were precipitated overnight at −20 °C in the presence of 5 vol of methanol containing 0.1 M ammonium acetate. The pellets recovered by centrifugation were rinsed 3 times with cold methanol and acetone, dried, and dissolved in the solubilization buffer. The supernatants from shoots and roots protein extracts were incubated with 5 mM tributyl phosphine and 10 mM acrylamide for 1 h at room temperature to reduce disulfide bonds and alkylate cysteine thiol groups. The reactions were stopped with 10 mM DTT, and the samples were stored at −80 °C. The protein concentration was determined using Bradford reagent (Bio-Rad).

Gel Image and Statistical Analyses

Gels were scanned using a Bio-Rad VersaDoc 1000 imaging system. The position of individual proteins on gels was evaluated with PDQuest software (version 7.3; Bio-Rad). The software program automatically carried out background subtraction. The gel image showing the higher number of spots and the best protein pattern was chosen as a reference template, and spots in a standard gel were then matched across all gels. Spot quantity values were normalized in each gel by dividing the raw quantity of each spot by the total quantity of all of the spots included in the standard gel. Gels were divided into two groups: 72 h mock-inoculated and 72 h inoculated for root and shoot, respectively. The comparisons performed were 72 h mock-inoculated root versus 72 h inoculated root and 72 h mock-inoculated shoot versus 72 h inoculated shoot. For each protein spot, the average spot quantity value and its variance coefficient in each group were determined. Global relationship among maps was analyzed by unsupervised principal component analysis (PCA) with Simca-P+ (Umetrics) using normalized spot volumes of individual gel replica. Normalized spot volumes were scaled to Unit Variance. Besides multivariate analysis, univariate Student’s t test analysis was performed to identify sets of proteins that showed a significant change (p < 0.01) with a fold change threshold ≥2. In-Gel Digestion and Peptide Sequencing by nanoHPLC-Chip-MS/MS

The pieces of gel containing the protein of interest were carefully excised from the stained gels. Protein spots were digested overnight at 37 °C using 12.5 ng/μL of sequencing grade modified porcine trypsin (Promega). The supernatant containing the extracted tryptic peptides was completely dried and redissolved in 5 μL of 2% acetonitrile and 0.1% formic acid before MS analysis. Peptides from each sample were separated by reversed phase nano-HPLC-Chip technology (Agilent Technologies) online-coupled with a 3D ion trap mass spectrometer (model Esquire 6000, Bruker Daltonics). The chip was composed of a Zorbax 300SB-C18 (43 mm × 75 μm, with a 5 μm particle size) analytical column and a Zorbax 300SB-C18 (40 nL, 5 μm) enrichment column. The complete system was fully controlled by ChemStation (Agilent Technologies) and EsquireControl (Bruker Daltonics) softwares. A sample volume of 5 μL was loaded onto the HPLCChip by the autosampler at a flow rate of 4 μL/min. Peptides were eluted sequentially at a flow rate of 300 nL/min, using a linear gradient from Solution A (2% acetonitrile; 0.1% formic acid) to 50% of Solution B (98% acetonitrile; 0.1% formic acid) in 20 min. The scan range used was from 300 to 1800 m/z. For tandem MS experiments, the system was operated with automatic switching between MS and MS/MS modes. The acquisition parameters for the instrument were ICC target, 30000; maximum accumulation time, 50 ms. Fragmentation options include SmartFrag (to ramp fragmentation energy for most efficient and reproducible MS/MS fragmentation);

Two-Dimensional Gel Electrophoresis

Total proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis (2-DE); 5 replica gels were performed loading 600 μg of proteins per gel. For the first dimension, isoelectric focusing (IEF) was carried out on 17 cm long pH 3− 10 NL IPG strips (Bio-Rad). Briefly, the strips were rehydrated for 8 h with 400 μL of solubilization buffer containing 1.5 mg/ mL of total protein extract. IEF was carried out with a Protean IEF Cell (Bio-Rad) applying a low initial linear voltage ramp (0−1000 V over 12 h) and then a rapid voltage ramp to 10,000 V with a limiting current of 50 μA/strip. After the IEF, IPGs strips were equilibrated in 6 M urea, 2% SDS, 20% glycerol, 375 mM Tris-HCl, pH 8.8 for 26 min. For the second dimension, SDS-PAGE was done using 8−18% acrylamide gradient gel and carried out with a Protean Plus Dodeca cell (Bio-Rad) in the 410



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Figure 1. Medicago truncatula response to Sinorhizobium meliloti infection. (A) Representative images of M. truncatula roots 72 h after S. meliloti inoculation. 1,2: presence of adhesion zones and different stages of root hair cells curling. 3: nodule primordium. (B) qRT-PCR analysis of MtN5 (left) and MtCLE1, MtCLE12, and MtCLE13 (right) mRNA levels in mock-inoculated and inoculated roots. The values reported are means ± standard error (SE) (n = 3). The data were analyzed by Student’s t test. * p < 0.05; ** p < 0.01 versus mock-inoculated.

CutOff, 27% of the m/z of the precursor ion; number of precursor ions, 3; threshold abs, 60000; width of isolation, 4 m/ z; fragmentation amplitude, 1.5 V; active exclusion after 2 spectra; release after 0.20 min. Advanced MS/MS parameters to define masses that shall be detected although smaller than base peak were Max Res Scan Avarages, 5; MS(n) avarages, 3; MS(n) isolation width, 4 m/z. The acquired spectra were processed in DataAnalysis 3.2 (Bruker Daltonics) to find compounds automatically, and deconvoluted spectra were submitted to Mascot search program (Matrix Sciences). Proteins were identified by searching in the National Center for Biotechnology Information nonredundant (NCBInr) viridiplantae protein database (NCBInr 20130918 database, 32611672 sequences; 11345269536 residues). For database searches, the following parameters were adopted: mass tolerance ± 0.9 Da, fragment mass tolerance ± 0.9 Da, missed cleavages 1. Propionamide formation on cysteine residues, oxidation of methionine residues, and acetylation of N-term peptide were specified as variable modifications. For positive identification, the score of the result of [−10 × Log P] had to be over the significance threshold level (p < 0.05).

cDNA was amplified using SYBR Green qPCR Supermix-UDG (Invitrogen) on the ABI Prism 7000 Sequence Detection System (Applied Biosystems). The qRT-PCR was performed using the following cycling conditions: 2 min at 50 °C, 2 min at 95 °C, 40 cycles of 95 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s and finally 72 °C for 2 min. All quantifications were normalized according to Maunoury et al.53 For each determination of mRNA levels, three cDNA samples derived from three independent RNA extractions were analyzed. For each amplification reaction, the analysis of the product dissociation curve was performed to exclude the presence of nonspecific amplifications (i.e., primer-dimer products). Data from qRTPCR experiments were analyzed according to the 2−ΔΔCt method.54 The list of primers adopted for qRT-PCR is reported in Supporting Information Figure S1. For MtN5 and MtCLE expression analyses the primer pairs employed were those already reported in Pii et al.52 and Mortier et al.,31 respectively.

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RESULTS

Experimental Setting and Phenotype of M. truncatula Inoculated Roots

Quantitative Real Time-PCR Analysis

To examine local and systemic proteomic changes at an early stage of rhizobial infection, we compared the root and shoot proteome of S. meliloti-inoculated M. truncatula plants with those of mock-inoculated ones at 72 h post inoculation (hpi). The sampling time point (72 hpi) was chosen based on phenotypic and molecular analyses. We performed microscopic observations on 3 independent sets of rhizobia-inoculated plants. In all of the S. meliloti-treated roots at 72 hpi, we

For the quantitative RT-PCR (qRT-PCR), we used a new set of plants. For each sample, 10 independently treated plants were used. Total RNA was isolated using the RNeasy mini kit (QIAGEN) starting from 100 mg of frozen pooled tissues and treated with RQ1 DNase (Promega). Comparative PCR analysis was carried out using first strand cDNA obtained with ImProm-II Reverse Transcription System (Promega). 411



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Figure 2. Representative 2-D gels of proteins extracted from mock-inoculated and rhizobia-inoculated plants. Maps from shoots and roots are green and gray boxed, respectively. For the first dimension, proteins were focused on 17 cm-long pH 3−10 NL IPG strips. For the second dimension, proteins were separated on SDS-PAGE using 8−18% acrylamide gradient gels (range 10−150 kDa).

Considering that CLE peptides are signaling molecules, which putatively interact with LRR-RLKs in the shoots, we have also monitored the expression levels of MtCLE1, MtCLE12, and MtCLE13 in the aerial parts of mock-inoculated and inoculated plants. We did not observe differences in MtCLE1 transcript level (Supporting Information Figure S2). Under our experimental conditions, MtCLE13 expression was undetectable, and MtCLE12 displayed only a limited and unchanged expression (Supporting Information Figure S2).

observed many adhesion zones and curling events (Figure 1, panel A1,2); nodule primordia were rare (from 0 to 2 per plant) (Figure 1, panel A3) while young nodules were absent. At an earlier stage of inoculation (18 hpi), few adhesion zones and curling events were visible (data not shown). At 7 days after inoculation, numerous primordia and young nodules were present in all of the plants (data not shown). To support the phenotypic observations, we evaluated at 72 hpi the mRNA level of MtN5 gene,52 a marker of early response to rhizobia implicated in epidermal stages of rhizobium−host interaction.55 The expression of MtN5 gene was 20-fold higher in inoculated roots compared with mock-inoculated (Figure 1B, left panel). In addition, we assayed the expression of three CLE genes, named MtCLE1, MtCLE12, and MtCLE13. MtCLE12 and MtCLE13 are symbiosis-related genes putatively involved in AON, whereas MtCLE1 is not induced after inoculation with S. meliloti.31 The induction of MtCLE13 occurs in the roots very early after infection, whereas MtCLE12 induction is restricted to young and mature nodules.31 In our experimental condition, the steady state level of MtCLE13 mRNA was 4-fold increased in inoculated roots as compared with mockinoculated ones (Figure 1B, right panel). We did not detect significant differences in the transcript levels of MtCLE1 and MtCLE12 (Figure 1B, right panel). These findings suggest we were monitoring the host responses occurring during preinfection/initial infection processes.

Changes in Root and Shoot Protein Profiles

Total protein extracts isolated from root and shoot tissues were separated on 2-DE gels and stained with Sypro Ruby. Protein patterns were analyzed using PDQuest software. We detected approximately 500 spots in each gel within a 3−10 pH range and a 10−150 kDa size range (Figure 2). Small proteins and peptides with a potential role in rhizobial symbiosis (e.g., CLE peptides and MtN5) cannot be revealed using this experimental procedure. The gel replicas were compared by multivariate and univariate statistical analyses. Variability of both protein data sets was examined by unsupervised principal component analysis (PCA) (Figure 3). Regarding the shoot protein data set, the first and second components explained the 21.06% and 19.51% overall variance, respectively (Figure 3A), while for the root protein spot data set, the first and second components explained the 20.92% and 17.57% overall variance, respectively (Figure 3B). Mock-inoculated and inoculated samples were 412



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Figure 3. Unsupervised PCA model of protein data sets. (A) Shoot PCA scatterplot representing 5 mock-inoculated and 5 inoculated observations. The two principal components PC1 and PC2 were 21.06% and 19.51%, respectively. (B) Root PCA scatterplot representing 5 mock-inoculated and 5 inoculated observations. The two principal components PC1 and PC2 were 20.92% and 17.57%, respectively. (■) mock-inoculated observations; (●) inoculated observations.

split into two different groups for both shoot and root protein data sets (Figure 3). This indicates that enough natural variation exists between the groups, increasing the confidence that interesting protein modulations can be found in these data sets. Student t test analysis between mock- and bacteria-inoculated plants revealed that 16 protein spots in the shoots and 19 in roots were differentially expressed (p < 0.01) with a fold change threshold ≥2. In particular, 12 spots were up-regulated and 4 down-regulated in the shoots, while 14 and 5 were up- and down-regulated in the roots, respectively. The percentage of differentially expressed protein was 3.2% and 3.8% in shoots and roots, respectively. The differentially expressed proteins are indicated by the corresponding spot number on the shoot and root standard maps reported in Figure 4, panels A and B, respectively.

Figure 4. Standard maps generated by PDQuest analyses. The 16 and 19 protein spots showing statistically significant differences (p < 0.01) in shoots (A) and roots (B) of mock-inoculated and rhizobiainoculated plants are marked by an open circle and numbered.

identified proteins, the experimental Mr reasonably agreed with the theoretical values of the matched proteins (Tables 1 and 2). Several proteins were represented in more than one spot presumably due to putative post-translational modifications, different splicing variants, separation of multimeric complexes, and proteolytic processing. Ten out of 13 proteins identified in the shoots and 16 out of 18 proteins in the roots corresponded, by Mascot analysis, to M. truncatula proteins with a putative function, whereas the remaining (1108, 2009, and 2111 gi|388511263 in Table 1; 306 and 5102 in Table 2) were classified either as protein of unknown function or as hypothetical proteins of M. truncatula. The analysis using the recently released MT4.0v1 database allowed the annotation of 4 of these proteins (Tables 1 and 2). In detail, 1108 can be annotated as a ribonucleoprotein (Medtr4g104470.1), 2009 as an oxygen-evolving enhancer protein 2 (Medtr3g449930.1), 2111 gi|388511263 as a GroES chaperonin (Medtr2g043230.1), and 5102 as a type II

Identification of Differentially Expressed Proteins

The identification of differentially expressed proteins based on raw MS/MS data was performed using Mascot program, searching against the NCBInr protein database. We identified 13 and 18 proteins from shoots and roots, respectively (Tables 1 and 2). The remaining differentially expressed proteins (4 for the shoot, i.e., spots 1003, 2003, 6003, 8001; 1 for the root, i.e., spot 3401) were not identified probably because of their relative low concentration. All M. truncatula identified proteins are listed in Tables 1 and 2 with the corresponding spot number, the NCBI accession number, the number of identified peptides, the Mascot score, the experimental and theoretical molecular mass (Mr) and isoelectric point (pI), protein sequence coverage, and the fold change variation (p < 0.01). In general, for most of the 413



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Table 1. List of Differentially Expressed Proteins Identified by Mass Spectrometry Analysis in M. truncatula Shoots 72 h after S. meliloti Infection spot number

protein name

201

14-3-3 protein

1007

ABA-responsive protein 17 ABR17

1108

87% identity Ribonucleoprotein (Medtr4g104470.1) Harpin binding protein

1109 2009 2111

91% identity Oxygen-evolving enhancer protein 2 (Medtr3g449930.1) 99% identity GroES chaperonin (Medtr2g043230.1) Ferritin-3

2602

Heat shock protein

4803

Phospholipase Dα

5004

Cysteine proteinase inhibitor

5703

Succinate dehydrogenase

7002

Armadillo repeat-containing protein

9013

PSI reaction center subunit II

NCBI accession no.

no. of peptides identifieda

Mascot scoreb

Mr (Da) exptl/ theor

pI exptl/ theor

sequence coverage (%)

fold variationc I vs mock-I

17

374

30000/29192

3.5/4.70

35

2.79

4

235

15000/16565

4/4.98

29

0.25

14

284

29000/33523

4.3/4.93

18

2.86

5

257

29000/29677

4.3/6.91

19

3.25

11

296

28000/28143

4.5/7.66

31

0.46

8

262

28500/27143

4.6/9.02

30

4.91

7

203

28500/28058

4.6/5.66

19

12

384

75000/72307

4.6/5.87

21

2.15

4

162

90000/91952

4.8/5.50

5

3.96

1

79

10000/11304

5.2/9.86

15

3.27

4

187

75000/68879

5/6.10

6

2.59

2

61

15000/87044

7.3/8.26

2

5.23

3

138

25000/23034

9.5/9.58

15

0.45

gi| 357489745 gi| 357449125 gi| 388508864 gi| 357441103 gi| 217072770 gi| 388511263 gi| 357468557 gi| 357476131 gi| 357455227 gi| 357458857 gi| 357483399 gi| 357495247 gi| 357480841

a Number of peptides identified: number of peptides assigned at p < 0.05. bMascot score: the protein score obtained by the sum of the highest ions score for each distinct sequence. cFold variation in spot abundance between rhizobia-inoculated (I) and mock-inoculated (mock-I) wt.

species (ROS) scavenging at this stage of rhizobial infection (Table 2). In addition, rhizobia-treated roots displayed a downaccumulation of a GTP-binding nuclear protein Ran-A1, involved in intracellular protein transport (5304) and of the enzyme methionine synthase (4810) (Table 2). The modifications observed in shoots upon radical infection were related to carbon metabolism, biotic stress, and hormone responses (Table 1). Six differentially expressed proteins (1108, 1109, 2009, 2111 gi|388511263, 2111 gi|357468557, and 9013) were predicted to be localized in the chloroplast.61 The two proteins (2009 and 9013) that are involved in the photosynthetic process were significantly reduced. Among the proteins implicated in regulating primary metabolism, a 14-3-3 protein (201), a kinase-like protein with a major role in nitrogen and carbon metabolism regulation, was induced in shoots of inoculated plants. 14-3-3 proteins control the tricarboxylic acid (TCA) cycle through interaction with its metabolic enzymes.62 In line with this observation, a succinate dehydrogenase, involved in both the TCA cycle and aerobic respiratory chain, was up-regulated in rhizobia-treated plants. A second group of differentially expressed proteins comprises those associated with biotic stress responses. A harpin binding protein (1109) and a cysteine proteinase inhibitor (5004; see Supporting Information Figure S3) were induced in treated plant, whereas abscisic acid (ABA)-responsive 17 (ABR17) protein (1007), a member of the pathogenesis-related proteins 10 (PR10) family, was reduced. In addition, the abundance of a ferritin-3 (2111 gi|357468557) was enhanced in response to rhizobia; this protein, involved in plant iron storage, is upregulated in response to bacterial infection.63

peroxiredoxin (Medtr2g022660.1). The 306 spot did not show similarity with any M. truncatula protein in MT4.0v1 database but displayed 45% identity with a glutamate-rich protein of Lotus japonicus (UniProt id. Q75QC2). In roots of inoculated plants, the most predominant changes concerned the induction of proteins involved in the carbohydrate and phenylpropanoid metabolisms (Table 2). Among the proteins assignable to carbohydrate metabolism, a fructose-bisphosphate aldolase (802, 5508, 5514), involved in the glycolytic pathway, and a sucrose synthase (5811, 5901, 6812, 6813), a key enzyme of sucrose metabolism essential for symbiotic nitrogen fixation,56−58 were resolved into multiple spots. In the case of fructose-bisphosphate aldolase (Table 2), protein spot 802 (∼80 kDa experimental Mr) probably corresponds to the dimeric form of the enzyme, whereas 5508 and 5514 (∼40 kDa experimental Mr) might represent the monomeric form.59 The presence of various forms of sucrose synthase is most likely due to post-translational modifications.60 A second group of differentially expressed proteins was represented by proteins associated with phenylpropanoid metabolism, such as isoflavone reductase-like protein (4405) and NAD(P)H-dependent 6′-deoxychalcone synthase (1405, 2408) that were detected. Flavonoids play a role during plant defense against pathogens, act as inducers of NFs biosynthesis, and affect hormonal balance. Two isoforms of alcohol dehydrogenase (307, 6507), an enzyme involved in several processes, including carbon degradation under oxygen-limiting conditions, cell-wall synthesis, and remodeling, were also found induced (Table 2). The reduction of proteins involved in counteracting oxidative stress (i.e., 2307, ascorbate peroxidase; 5102, peroxiredoxin) might indicate a decreased need in mechanisms of reactive oxygen 414



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Table 2. List of Differentially Expressed Proteins Identified by Mass Spectrometry Analysis in M. truncatula Roots 72 h after S. meliloti Infection spot number

protein name

306

Hypothetical proteina

307

Alcohol dehydrogenase

802

Fructose-bisphosphate aldolase

1405

NAD(P)H-dependent 6′deoxychalcone synthase Cytosolic ascorbate peroxidase

2307 2408 4405

NAD(P)H-dependent 6′deoxychalcone synthase Isoflavone reductase-like protein

4515

Alcohol dehydrogenase

4810

Methionine synthase

5102

5508

93% identity Type II peroxiredoxin (Medtr2g022660.1) GTP-binding nuclear protein Ran-A1 (Fgm)e Fructose-bisphosphate aldolase

5514

Fructose-bisphosphate aldolase

5811 5901 6507

Sucrose synthase Sucrose synthase Alcohol dehydrogenase

6812 6813

Sucrose synthase Sucrose synthase

5304

NCBI accession no.

no. of peptides identifiedb

Mascot scorec

Mr (Da) exptl/ theor

pI exptl/ theor

sequence coverage (%)

fold variationd I vs mock-I

gi| 357480473 gi| 357463695 gi| 357490465 gi| 357462577 gi| 357472451 gi| 357462577 gi| 357483525 gi| 357463695 gi| 357508777 gi| 217071078 gi| 357466305 gi| 357490465 gi| 357490465 gi|4584692 gi|4584692 gi| 357463695 gi|4584692 gi|4584692

9

190

34000/15048

3.5/3.90

35

10.44

2

90

30000/41071

3.5/5.97

5

13.96

5

123

85000/78302

4/5.76

6

2.52

3

110

35000/35137

5/5.72

10

3.88

6

160

30000/27137

5.5/5.52

20

0.52

8

296

35000/35137

5.5/5.72

37

2.14

10

314

36000/33798

6/5.62

30

5.31

11

227

40000/41071

5.9/5.97

17

2.16

7

206

85000/88795

6/6.16

6

0.31

2

102

20000/21478

6.5/8.43

15

0.34

3

107

33000/88590

7/6.63

4

0.45

11

383

41000/78302

7/5.76

10

2.64

10

132

41000/78302

6.5/5.76

2

2.47

13 19 14

304 585 414

90000/92278 90000/92278 41000/41071

7.2/5.86 7/5.86 7.3/5.97

11 16 23

19.98 2.75 48.56

10 19

309 633

90000/92278 90000/92278

7.3/5.86 7.3/5.86

11 20

20.08 25.75

a

45% identity with glutamate-rich protein (Lotus japonicus, Q75QC2). bNumber of peptides identified: number of peptides assigned at p < 0.05. Mascot score: the protein score obtained by the sum of the highest ions score for each distinct sequence. dFold variation in spot abundance between rhizobia-inoculated (I) and mock-inoculated (mock-I) wt. eFgm = fragmented. c

(e.g., photosynthetic reactions, TCA cycle, and protein modifications). The transcript level of 14-3-3 protein (201), harpin binding protein (1109), GroES chaperonin (2111 gi| 388511263), ferritin-3 (2111 gi|357468557), phospholipase D α (4803), and cysteine protease inhibitor (5004) increased at 72 hpi in the shoots of rhizobia-inoculated plants, confirming the proteomic data (Figure 5C−H). The 1007 protein was 75% reduced in the shoots of rhizobia-inoculated plants versus mock-inoculated, whereas the expression level of the transcript did not significantly change at this time point (Figure 5I). Therefore, we have conducted the analysis at earlier time points (3, 12, 18 hpi). The transcript abundance of 1007 gene was significantly reduced at 3 hpi compared with its level in mockinoculated plants (Figure 5I). Thus, the discrepancy between transcript and protein pattern might depend either on protein stability or on a delayed translation. The expression pattern of two differentially expressed proteins, 5901 (sucrose synthase) and 201 (14-3-3 protein) was also assayed in M. truncatula NODULE INCEPTION (NIN) mutant. Excessive root hair curling, impaired infection and inhibition of nodule formation characterize the Mtnin mutant.67,68 NIN protein functions downstream of the early Nod Factors signaling pathway to coordinate nodule organogenesis and bacterial entry.67,68 The induction of 201 gene expression observed in wild-type rhizobia-inoculated plants was greatly reduced in Mtnin mutant, whereas the induction of 5901

The systemic response of rhizobia-infected plants included also the induction of a heat-shock protein 70 kDa (2602) and a GroES chaperonin (2111 gi|388511263), molecules that assist protein folding also under stress condition. We additionally observed the accumulation of phospholipase D (PLD) (4803) upon rhizobial infection. PLD is involved in many plant cellular processes, such as signaling in stress and hormone responses.64−66 Validation of Proteomic Data by Quantitative RT-PCR Analysis

To obtain an independent confirmation of the proteomic data, we performed qRT-PCR using new sets of plants. We have focused the analyses on shoot proteins, since most of those identified in the roots have already been associated to nodulation and N-fixation processes. The expression patterns of 9 differentially accumulated proteins, 2 in the root and 7 in the shoot, were analyzed. In roots at 72 hpi, the changes in transcript levels of peroxiredoxin (5102) and sucrose synthase ( 5901) mirrored the proteomic data; 5102 and 5901 mRNA abundances were 28% reduced and 71% induced, respectively, in rhizobia-inoculated roots compared with mock-inoculated ones (Figure 5A,B). Among the 13 differentially expressed shoot proteins, 7 proteins, involved in either biotic stress response or hormone signaling and metabolism, were chosen for validation. The remaining 6 are proteins playing a role in general metabolism 415



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Figure 5. Validation of differentially expressed proteins by qRT-PCR analyses. (A,B) The mRNA levels of 5102 and 5901 were evaluated after 72 h of treatment in mock-inoculated and inoculated roots . (C−I) The mRNA levels of 201, 1109, 2111, 4803, and 5004 were evaluated after 72 h of treatment in shoots of mock-inoculated and inoculated plants. The mRNA levels of 1007 (I) was evaluated along a time course of inoculation (3, 12, 18, and 72 hpi). The values reported are means ± standard error (SE) (n = 3). Student’s t test was applied. *p < 0.05; **p < 0.01; ***p < 0.001 versus the respective wt mock-inoculated.

“omics” approach used, these investigations generally pertain to molecular events taking place in the regions of the root where rhizobium is hosted, whereas the response of the whole root has been poorly considered. The transcriptional changes occurring in the shoots of M. truncatula nodulated plants in response to nitrogen status have been described by Ruffel et al.,72 but as far as we know, the systemic response to S. meliloti has never been assessed through proteomics at any stage of the symbiosis. The occurrence of a systemic response in endosymbiotic interaction is proved also by shoot proteome and

gene was abolished (Supporting Information Figure S4). These data suggest that the changes in the proteins’ abundance are symbiosis-related responses.

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DISCUSSION Transcriptomics has largely been applied to decipher the molecular events that characterize the symbiotic interaction between Medicago truncatula and the N-fixing bacterium Sinorhizobium meliloti,43,53,69 while only a few proteomic studies have been carried out.38,41,70,71 Independently of the 416



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transcriptome changes detected in M. truncatula in response to mycorrhizae.73,74 On these premises, we have addressed the proteome changes occurring in the shoot and in the whole root of M. truncatula plants inoculated with S. meliloti. The aim of this study was to investigate whether a systemic response is already triggered at an early stage of rhizobial infection, since the AON, which requires a systemic signaling, occurs after the formation of the first nodule primordia.25 The proteomic analysis was conducted on plants showing deformation and curling of the root hairs and only in rare cases incipient nodule primordia. This phenotypic observation was supported by the increased expression of the MtN5 gene, an early marker of rhizobial infection.55 We have also analyzed two genes that code for CLE peptides acting as signaling molecules of AON.25,31 MtCLE12 and MtCLE13 are both induced during rhizobial symbiosis, but MtCLE13 more precociously than MtCLE12.31 In the roots collected for the proteome analysis MtCLE13 was already induced, whereas the expression of MtCLE12 is unaltered, suggesting a systemic response to rhizobium might already be triggered. The separation of the extracted proteins by 2-DE and Sypro Ruby staining allowed the detection of about 500 proteins in both shoots and roots, values comparable with those reported in other proteome analyses on various M. truncatula organs.39,75 In plants treated with S. meliloti approximately 3% of the proteins separated by 2-DE were differentially expressed in both shoots and roots (17 and 19 proteins, respectively). A similar number of proteins resulted differentially accumulated in shoots of mycorrhizal plants in comparison to those grown in the absence of the fungus.73 A few differentially expressed proteins were also detected upon rhizobial infection when the entire root was analyzed.71

induction of alcohol dehydrogenase has been observed only in immature and mature nodules.53 Our data indicate that alcohol dehydrogenase is also strongly induced before nodule formation. Among the down-regulated proteins, a cytosolic ascorbate peroxidase and a type II peroxiredoxin were detected. Maunuory et al.53 found decreased transcript levels of ascorbate peroxidase during the formation of the root nodules, and peroxiredoxin expression was documented in root nodules of Lotus japonicus and other legumes.77 Both ascorbate peroxidase and peroxiredoxin are implicated in the protection against oxidative processes under biotic and abiotic stress conditions. Their reduced abundance might be related to a general decline of the defense response against rhizobia. Several transcriptomic studies have revealed a transitory induction of defensive genes upon rhizobial inoculation, followed by a phase of suppression of stress-responsive genes.43 The increased flavonoid biosynthesis at very early phases of rhizobial and arbuscular mychorrizal symbiosis has been documented for M. truncatula roots.41,43 The accumulation of enzymes of the flavonoids biosynthetic pathway (i.e., two isoforms of NAD(P)H-dependent 6′-deoxychalcone synthase and an isoflavone reductase-like protein) was observed also in our study. The increase in flavonoids synthesis is commonly referred to as bioprotective activity; however, some pathway intermediates act as elicitors of NFs biosynthesis in rhizobia. In addition, root flavonoids affect auxin distribution in rhizobiatreated roots, contributing to nodule primordia formation.78 Lohar et al. reported that the pattern of disease response genes and flavonoid biosynthesis genes have opposite expression profiles during rhizobial infection, suggesting other roles for flavonoids during symbiosis in addition to antimicrobial activity.43 The enzyme methionine synthase and the GTP-binding nuclear protein Ran-A1 (Ras-related nuclear protein-A1) are described here for the first time as associated to S .meliloti infection. Both proteins were down-regulated in roots treated with the rhizobium. The enzyme methionine synthase is involved in the production of methionine, which is incorporated into proteins or acts as an intermediate in the synthesis of S-adenosylmethionine (SAM) and ethylene. Ethylene is implicated in the control of nodule number and regulates the position of the nodules opposite to xylem poles.79 Changes in the methionine biosynthesis can affect SAM production and consequently can modulate the levels of ethylene in the root. Ran-A1 is a GTP-binding protein belonging to the subfamily Ran, whose members are involved in nuclear-cytoplasmic transport. GTP-binding proteins function as signaling molecules in many biological processes such as protein trafficking, stress, signal transduction pathways, and cell division.80 Some GTP-binding proteins are specifically expressed in the root nodules,81 and a Rac1 GTPase of M. truncatula (i.e., MtROP9) is implicated in early infection signaling.82 However, it remains to be clarified whether the identified form of GTP-binding nuclear protein Ran-A1 (which has an experimental Mr lower than theoretical, 5304 in Table 2) represents an alternative splice variant or a cleaved form derived from endogenous proteolysis.

Root Proteome Analysis

Eighteen differentially expressed proteins were identified by MS/MS. The most relevant effect observed in the root proteome was the accumulation of proteins involved in carbohydrate metabolism, in particular those associated with C-consuming processes, such as sucrose synthase, fructosebisphosphate aldolase, and alcohol dehydrogenase. Sucrose synthase 1 (MtSucS1) is the principal enzyme responsible for sucrose degradation in mature nodules, and its down-regulation caused impaired nodulation and reduced Nfixation.76 The role of SucS1 in the root nodule has been associated with the increased requirement of metabolic energy for N-fixation and for generation of C skeleton for NH4+ assimilation. In this work, we show for the first time that MtSucS1 is accumulated very early after rhizobial infection, suggesting the root increases its sink activity well before the onset of N-fixation in the nodules. MtSucS1 is crucial also for the establishment and maintenance of arbuscules in the arbuscular mycorrhizal symbiosis.76 The presence of different SucS1 forms in rhizobia-inoculated roots probably reflects the existence of post-translational modification sites within the sequence. The N-terminal region of the SucS1 protein presents several seryl residues that can be phosphorylated.60 Phosphorylated SucS1 isoforms were found in M. truncatula nodules, although the physiological meaning of these modifications is still not elucidated.60 The early induction of fructose-bisphosphate aldolase in roots treated with S. meliloti has been documented in other transcriptomic and proteomic surveys,41,43 whereas the

Shoot Proteome Analysis

This work explored for the first time the changes that are triggered systemically in M. truncatula shoot proteome under rhizobial symbiosis. We demonstrated that a systemic response 417



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out of 13 differentially expressed proteins are localized within the chloroplast, indicating that chloroplast metabolism in general was altered in response to rhizobial infection.

occurred at a very early stage of rhizobial infection before the formation of the nodule primordia. These data were extensively confirmed through the analysis at the transcript level of the principal differentially expressed proteins. In contrast to protein changes in the roots, the modifications in the shoots were generally modest in the order of 2−5 times. Some of these proteins or their mRNAs (e.g., cysteine protease inhibitor, HSP70, PLD, ferritin-3, a 14-3-3 protein) are differentially expressed also in roots colonized by rhizobium.43,53,71 Many proteins involved in defense responses or abiotic stresses (i.e., harpin binding protein, cysteine protease inhibitor, heath shock protein) were systemically induced after rhizobial infection. In particular, the harpin binding protein of M. truncatula exhibits a high identity (74%) with the A. thaliana fibrillin 4 (FBN4/FIB4) protein (At3g23400). In A. thaliana, the mutation of FBN4/FIB4 resulted in an increased susceptibility to pathogenic bacteria and the expression of the FBN4/FIB4 was induced by lipopolysaccharides in tobacco suspension cells.83 We also observed an increased accumulation of the enzyme succinate dehydrogenase (SDH) that plays a central role in mitochondrial metabolism as a component of both TCA cycle and the electron transport chain. In addition, SDH is implicated in resistance against pathogens since SDH mutants displayed reduced mitochondrial ROS production and exhibited increased susceptibility to bacterial and fungal pathogens.84 The induction of defense-associated genes was also reported in the shoots of M. truncatula upon mycorrhization.74 Liu et al. suggested that arbuscular mycorrhizal symbiosis might trigger an induced systemic resistance (ISR) response to leaf pathogens.74 The ABR17 protein showed an opposite expression pattern compared to the other defense-related proteins. This PR10 protein is regulated by the phytohormone ABA, which has a repressive action on nodulation. According to the data available in the gene expression atlas of M. truncatula, ABR17 gene (Probeset Mtr. 10317.1.S1) is repressed in the root during nodulation.85 Other ABA-responsive proteins are transcriptionally down-regulated during rhizobial infection and nodulation.43,69 In this regards, PLD α, which is accumulated in the shoots in response to rhizobium, is involved in the signaling for plant hormones, particularly ABA.65,86 The ratio of ABA and cytokinin is relevant in root-shoot signaling and AON.87 Therefore, it would be very interesting to investigate the possible role of ABR17 and PLD in the systemic response to rhizobia. Another regulatory protein systemically induced by symbiosis is a 14-3-3 protein. The 14-3-3 proteins play important regulatory roles in numerous processes, such as carbon and nitrogen metabolism. Among the 14-3-3 metabolism-related targets, enzymes of Calvin’s cycle and glycolytic pathway as well as sucrose synthase, invertase, nitrate reductase, and glutamine synthetase are included.88,89 Recently, it was demonstrated that 14-3-3 proteins regulate the ethylene biosynthesis through stabilization of 1-aminocyclopropane-1-carboxylate synthase.90 In this view, the increased abundance of 14-3-3 protein might result in promotion of ethylene biosynthesis, suggesting that ethylene not only regulates nodule development locally but also participates in systemic signaling. Shoot carbon metabolism might be affected by the changes of proteins associated to photosynthetic pathways. Indeed, 6

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CONCLUSIONS Our work describes for the first time the occurrence of a systemic response to rhizobia at an early stage of infection. One of the changes observed in the shoots concerns the activation of defense-related proteins. A transient induction of genes involved in pathogen defense is a common feature in the early phases of root response to rhizobia. In addition, a systemic protection against pathogen attacks, a mechanism known as ISR, has been observed in some cases after root colonization by nonpathogenic rhizobacteria and mychorrizae.74 Further research should be addressed to investigate whether these biotic stress-related proteins are implicated in the control of nodulation. The changes in carbon metabolism emerge as another important issue in both roots and shoots, indicating that modifications of translocation and utilization of photosynthates are linked not only to the energy requirement during nodule development and N-fixation but to the initial phases of infection as well. Our data raise the possibility that the phytohormones ABA and ethylene participate in root-shoot communication during symbiosis. It is conceivable that sugar signaling plays an important role in rhizobial symbiosis and that the interplay between sugars and hormones is necessary to coordinate local and systemic responses to rhizobia. Altogether, the set of proteins identified in this work represents a good basis for deepening the current knowledge on the coordinated response of roots and shoots during rhizobial symbiosis.

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ASSOCIATED CONTENT

* Supporting Information S

Supplementary figures as referenced in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +39 045 8027918. Fax: +39 045 8027929. E-mail: tiziana. pandolfi[email protected]. Author Contributions †

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Pascal Gamas for kindly providing us with S. meliloti harboring pXLGD4 plasmid and Giles Oldroyd for M. truncatula nin mutant. We are grateful to Anita Zamboni for help with statistical analysis.

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REFERENCES

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