Differential gel electrophoresis (DIGE) to quantitatively monitor early symbiosis- and pathogenesis-induced changes of the Medicago truncatula root proteome

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Differential gel electrophoresis (DIGE) to quantitatively monitor early symbiosis- and pathogenesis-induced changes of the Medicago truncatula root proteome Leif Schenkluhn a , Natalija Hohnjec c , Karsten Niehaus a , Udo Schmitz b , Frank Colditz b,⁎ a

University of Bielefeld, Dept. 7, Proteome and Metabolome Research, Universitätsstraße 25, D-33615 Bielefeld, Germany Leibniz University of Hannover, Institute for Plant Genetics, Dept. III, Plant Molecular Biology/Plant Proteomics, Herrenhäuser Str. 2, D-30419 Hannover, Germany c Leibniz University of Hannover, Institute for Plant Genetics, Dept. IV, Plant Genomics, Herrenhäuser Str. 2, D-30419 Hannover, Germany b

AR TIC LE I N FO

ABS TR ACT

Article history:

Symbiosis- and pathogenesis-related early protein induction patterns in the model legume

Received 15 September 2009

Medicago truncatula were analysed with two-dimensional differential gel electrophoresis.

Accepted 23 October 2009

Two symbiotic soil microorganisms (Glomus intraradices, Sinorhizobium meliloti) were used in single infections and in combination with a secondary pathogenic infection by the oomycete

Keywords:

Aphanomyces euteiches. Proteomic analyses performed 6 and 24 h after inoculations led to

Arbuscular mycorrhiza (AM)

identification of 87 differentially induced proteins which likely represent the M. truncatula

Aphanomyces

root ‘interactome’. A set of proteins involved in a primary antioxidant defense reaction was

Differential gel electrophoresis

detected during all associations investigated. Symbiosis-related protein induction includes

(DIGE)

a typical factor of early symbiosis-specific signalling (CaM-2), two Ran-binding proteins of

Interactome

nucleocytoplasmic signalling, and a set of energy-related enzymes together with proteins

Medicago

involved in symbiosis-initiated C- and N-fixation. Pathogen-associated protein induction

Sinorhizobium

consists of mainly PR proteins, Kunitz-type proteinase inhibitors, a lectin, and proteins related to primary carbohydrate metabolism and phytoalexin synthesis. Absence of PR proteins and decreased pathogen-induced protein patterns during mixed symbiotic and pathogenic infections indicate bioprotective effects due to symbiotic co-infection. Several 14-3-3 proteins were found as predominant proteins during mixed infections. With respect to hormone-regulation, A. euteiches infection led to induction of ABA-related pathways, while auxin-related pathways are induced during symbiosis. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

In associations between plants and soil microorganisms, only symbiotic interactions have beneficial effects for the host plants while pathogenic interactions lead to severe damages.

The vast majority of land plants are capable of installing mutualistic symbioses with arbuscular mycorrhizal (AM) fungi, which are perhaps the most widespread but certainly significant associations established over 400 million years ago [1]. In contrast, the host range for an additional symbiosis with

Abbreviations: Ae, A. euteiches-infected; AM, arbuscular mycorrhiza; hpi, hours post inoculation; HR, hypersensitive response; MtGI, (DFCI) Medicago truncatula Gene Index (v9.0, at Dana-Farber Cancer Institute, Harvard School of Public Health, Boston, U.S.A.); myc, mycorrhized; nod, nodulated; PMF, peptide mass fingerprinting; PR proteins, pathogenesis-related proteins; ROS, reactive oxygen species; TC, tentative consensus sequence. ⁎ Corresponding author. Institute for Plant Genetics, Dept. III, Plant Molecular Biology/Proteomics, Leibniz University of Hannover, Herrenhäuser Str. 2, D-30419 Hannover, Germany. Tel.: +49 511 762 3603; fax: + 49 511 762 3608. E-mail address: [email protected] (F. Colditz). 1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.10.009

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Table 1 – Protein induction in Medicago truncatula roots after pathogenic (A. euteiches-) infection.

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phylogenetically diverse nitrogen-fixing rhizobial bacteria is mainly limited to legumes (Fabales) of the eurosids (I) orders forming the nitrogen-fixing clade [2,3]. The legumes acquire through mycorrhiza the macronutrients phosphorus, nitrogen, as well as most likely an array of micronutrients and from interactions with rhizobial bacteria reduced nitrogen due to the conversion of N2 into NH3 by bacterial nitrogenases [4]. Although these two symbiotic associations are quite different, they are accompanied by overlapping mechanisms in the host plant, like the transcriptional activation of a common subset of genes and the formation of an intracellular microbe–plant interface for nutritional exchange [5]. The first steps of these associations are signalling processes, prior to a physical contact between symbionts: flavanoids exuded by legume roots are perceived by rhizobial bacteria and induce expression of bacterial nodulation (nod) genes leading to the production of lipochitooligosaccharide (LPS) signal known as Nod factors. The sesquiterpene strigolactone induces mycelial branching in mycorrhizal hyphae and diffusible Myc factors are able to activate host mycorrhizal gene induction and promote lateral root formation [4,6,7]. Regarding the microcosm of plant–pathogen interactions, there coincidently exist compounds released by the microorganisms called general elicitors or pathogen-associated molecular patterns (PAMPs) that induce the first host signalling responses [8,9]. By contrast, these signalling pathways are assumed to be different from those of symbiotic interactions. However, the host response mechanisms during prepenetration and initial penetration processes are supposed to be very similar in symbiotic and pathogenic fungal interactions [10]. For pathogenic fungi, it is known that they trigger a so-called cytoplasmic aggregation of plant epidermal cells, which is a cytoskeleton-driven accumulation of organelles including the nucleus at the penetration side, while for AM associations, a so-called pre-penetration apparatus (PPA) in the form of a cytoplasmic column traversing the cell in a root-centripetal direction, was identified [11–13]. It is very likely, that – as an initial molecular response – the host cells induce a set of basal defense mechanisms. These include the generation of reactive oxygen species (ROS) during an oxidative burst, tightly embedded in a cellular hypersensitive response (HR) that restricts the ingress of the infecting organisms. Coevally, several antioxidant enzymes are pro-

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duced acting as interceptors to protect the cell from oxidative damage. In case of pathogenic challenges, secondary defense pathways and therewith complex molecular events are initiated as a result of a pronounced oxidative burst and HR reactions, mainly represented by defense-induced proteins (e.g. resistance (R-) proteins, pathogenesis-related (PR-) proteins) and antimicrobial compounds of secondary metabolism (e.g. phenylpropanoids, phytoalexins) [14]. During symbiotic associations, it was documented for Sinorhizobium meliloti infections that LPS fragments (lipid A) effectively suppress the initial oxidative burst in host plants, thus leading to a suppressive effect on subsequent defense-associated gene expression [15,16]. For an early AM symbiosis, results described in the literature are quite diverse, but there is evidence that a moderate oxidative burst is induced, accompanied by a transient induction of host plant defense genes during initial infection [17–20]. So far it is not known how AM overcomes this initial host defense and if the defense-related molecular pathways are strictly discrete from symbiosis-related ones. For the monitoring of early molecular host responses after symbiotic infections in comparison to pathogenic infections, we have carried out a two-dimensional differential gel electrophoresis (2D-DIGE) approach to identify protein induction in roots of the model legume Medicago truncatula after inoculation either with symbiotic microorganisms, represented by the AM fungi Glomus intraradices and the rhizobia bacterium Sinorhizobium meliloti, or with the pathogenic oomycete root pathogen Aphanomyces euteiches. In addition, we investigated changes in host responses during mixed infections with primary symbiotic and secondary pathogenic inoculation. A. euteiches is accountable for a severe root rot disease in several legume crop plants including pea as major host, where it is considered to be the most destructive disease in areas with temperate or humid climate [21–23]. The pathogenic interaction to M. truncatula was comprehensively investigated, mainly at the proteome level [24–26]. Very recently, initial molecular responses were identified by the establishment of an inoculation system for M. truncatula cell suspension cultures [27]. All microbial interaction partners investigated in this study exhibit a biotrophic infection phase that presupposes a living host. Moreover, the initial host responses appear to be very similar for symbiotic and pathogenic infections. Thus, identification of a distinct

Notes to table Protein number. b Best matching tentative consensus (TC) sequence identifier in the M. truncatula Gene Index (MtGI).cProtein encoded by the best matching gene. d Molecular weight (MW) and isoelectric point (pI), calculated from the position on the 2-DE gel. e Percentage sequence coverage of the PMF match, number of matching peptides. f Protein Regulation at 6 hours and 24 hours after inoculation:Ae = A. euteiches-induced; Myc = Mycorrhiza fungi G. intraradices-induced; Nod = induced after root nodulation with S. meliloti; MycAe/NodAe = induced in mycorrhized or nodulated roots secondarily infected with A. euteiches; Con = induced in control roots. Abundance index (Abun.), representing relative in-gel abundances:− = no detectable differences in abundance or even absence of protein; + = abundance is slightly increased (1.5- to 2-fold); ++ = increased abundance (2- to 2.5-fold); +++ = abundance is highly increased (> 2.5-fold).(SD value >0.5) = standard deviation (SD) is noted in brackets when value exceeds SD of ± 0.5. Protein regulation is divided into classes as highlighted with different colours: — Protein induction is specific for A. euteiches inoculation. — Protein induction is predominant after A. euteiches inoculation, but also appears during symbiotic or mixed inoculations or in controls. — Protein induction is specific for symbiotic inoculation. a

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molecular signalling specific for each type of interaction represents the major goal of our studies. Furthermore, changes in the protein profiles for mixed pathogenic and symbiotic infections may allow the detection of proteins that mediate beneficial effects for the plant by symbionts and even

“bioprotective” effects towards pathogenic infections resulting from co-infections with symbionts. The DIGE technology is supposed to provide the adequate experimental setting, since it allows separation of two protein samples on the same gel, thus eliminating gel-to-gel variability, which facilitates

Table 2 – Protein induction in Medicago truncatula roots during symbiosis (G. intraradices, S. meliloti).

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detection of changes in protein patterns and is especially effective to distinguish very small differences in migration occurring at the pI or Mr level that are hardly detectable via classical 2-DE resolution.

plants of each inoculation were harvested. Roots were immediately cut off, rinsed with sterilized water to remove Fahraeus medium and subsequently frozen in liquid nitrogen.

2.3.

2.

Materials and methods

2.1.

Plant material and inoculation

Seeds of M. truncatula (‘Jemalong A17’) were sterilized in concentrated sulphuric acid and sodium hypochlorite (3% v/v) each for 5 min, rinsed several times with sterilized water and germinated for 3 days in the dark at 24 °C. After germination, the seedlings were cultured in square Petri dishes (12 × 12 cm) on solid nitrogen-free Fahraeus medium [28] (24 ± 2 °C day (16 h), 18 ± 2 °C night (8 h); 60 ± 15% relative humidity). Culture plates were half covered with aluminum foil to reduce exposure of root areas to light. Inoculation of plant roots with 3 different soil microorganisms was carried out 7 days after transplanting by carefully applying a same volume of 0.5 ml of each inoculum to one entire plant root: (i) a mycorrhiza fungi G. intraradices spores inoculum containing 5.000 viable spores (Premier Tech, Rivière-du-Loup, Canada), (ii) a S. meliloti 1021 inoculum of a mid-log phase culture, and (iii) an A. euteiches strain ATCC 201684 (Rosendahl, Dept. of Mycology, Botanical Institute, University of Copenhagen, Denmark) zoospores inoculum containing 100,000 spores. Induction of zoospores was initiated as described before [25,27]. Inoculation was performed separately with one microorganism specie for one plant Petri dish in case of single infections. To obtain mixed infections, plant roots inoculated with either G. intraradices or S. meliloti as primary inoculum were secondarily infected with A. euteiches 3 h after the first infection step. For controls, 0.5 ml of sterilized water was applied to each plant root. Inoculation of M. truncatula roots was performed in four different experiments (four biological controls).

2.2.

Harvest of plants

Inoculated plants and control plants were harvested at two time-points: (a) 6 h, and (b) 24 h after infection. At least 50

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Total root protein extraction with phenol

About 0.5 g of root tissue was ground briefly in liquid nitrogen and homogenized in 750 µl lysis buffer (700 mM sucrose, 500 mM Tris, 50 mM EDTA, 100 mM KCl, 2 mM PMSF and 2% (v/v) ß-mercaptoethanol, pH 8.0). Protein extraction was performed according to a protocol of Hurkman and Tanaka [68] as described previously [25].

2.4.

Sample preparation for DIGE

100 µg of each protein sample was used for labelling reaction with CyDye™ Fluor minimal labelling reagents (GE Healthcare, Freiburg, Germany). Therefore, protein samples were resuspended in a minimum volume of CyDye labelling-compatible lyses buffer containing 40 mM Tris–HCl, 8 M urea, 4% (w/v) CHAPS at pH 8.5. Protein samples were labelled for two experimental approaches as following: (i) Cy2™ — control sample, (single inoculations:) Cy3™ — G. intraradices inoculation sample, Cy5™ — S. meliloti inoculation sample; (ii) Cy2™ — A. euteiches inoculation sample, (mixed inoculations:) Cy3™ — G. intraradices and A. euteiches coupled inoculation, Cy5™ — S. meliloti and A. euteiches coupled inoculation. Labelling reactions were performed according to the DIGE minimal labelling protocol. The Cy2™, Cy3™ and Cy5™ labelled samples for each experiment were mixed together and volume was adjusted to 350 µl with a rehydration buffer obtaining a final sample solution composed of 8 M urea, 2% (w/v) CHAPS, 0.5% DTT, 0.5% IPG buffer 3–10 NL (GE Healthcare, Freiburg, Germany). CyDye-labelled protein samples were analysed by 2-DE. A 1:1:1 mixture of all the three samples of each experimental approach (i, ii) was labelled with one CyDye and used as internal standard during gel electrophoresis to allow gel-to-gel matching and in-gel analyses of relative protein spot intensities. For each biological experiment carried out, the internal standard sample was used in substitution for one Cydyelabelled sample, so that three additional gels were carried out for analyses of protein intensities. Corresponding in-gel intensities for one protein analysed were averaged after

Notes to table Protein number. b Best matching tentative consensus (TC) sequence identifier in the M. truncatula Gene Index (MtGI). c Protein encoded by the best matching gene. d Molecular weight (MW) and isoelectric point (pI), calculated from the position on the 2-DE gel. e Percentage sequence coverage of the PMF match, number of matching peptides. f Protein regulation at 6 h and 24 h after inoculation:Myc = Mycorrhiza fungi G. intraradices-induced; Nod = induced after root nodulation with S. meliloti; MycAe/NodAe = induced in mycorrhized or nodulated roots secondarily infected with A. euteiches; Con = induced in control roots. Abundance index (Abun.), representing relative in-gel abundances:− = no detectable differences in abundance or even absence of protein; + = abundance is slightly increased (1.5- to 2-fold); ++ = increased abundance (2- to 2.5-fold); +++ = abundance is highly increased (> 2.5-fold).(SD value >0.5) = standard deviation (SD) is noted in brackets when value exceeds SD of ± 0.5. Protein regulation is divided into classes as highlighted with different colours: — Protein induction is specific for symbiotic inoculation. — Protein induction is predominant for symbiotic inoculation, but also appears during mixed inoculations or in controls. — Protein induction is specific for control Mock inoculation. a

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background subtraction, allocated with the internal standard, and standard deviation (SD) was determined as shown in Tables 1 and 2.

2.5.

2-DE

Two-dimensional gel electrophoresis was performed for four independent biological experiments at the two mentioned time-points of harvesting, by combining IEF (pI 3–10 NL) using an IPGphor system (Amersham Pharmacia Biotech, Uppsala, Sweden) with a sodium dodecyl sulphate-tricine gel electrophoresis as described previously [25]. Additionally, three technical repetitions were performed for each biological experiment. For protein identification by MS analysis, additional gels were prepared with the same sample composition as described for the DIGE samples but using 300 µg of each protein sample reaching a final protein concentration of 900 µg for mixed samples. Gels were Coomassie-stained overnight with 0.1% Coomassie brilliant blue CBB-G250 as described previously [25].

2.6.

Image acquisition and analysis

After electrophoretic separation, gels were scanned on a Typhoon Trio Fluorescence Scanner (GE Healthcare) at 100 micron resolution using the appropriate excitation wavelengths: 488 nm (Cy2™), 532 nm (Cy3™) and 633 nm (Cy5™). Photo-digitalized gel images were visualized with the Image Quant v5.2 analysis software and evaluation of in-gel relative spot intensities of protein patterns was analysed employing the DeCyder v5.01 software (GE Healthcare) with the components DIA (differential in-gel analysis) and BVA (Biological Variation Analysis) for three gels of each biological experiment carried out.

2.7. Tryptic in-gel digestion of proteins and MALDI-TOF/ mass spectrometry-based protein identification Protein spots of interest were excised manually from the Coomassie-stained gels using a GelPal Protein Excision System (Genetix, Queensway, U.K.) and in-gel digestion with trypsin (sequencing grade modified trypsin; Promega, Madison, WI, U. S.A.) was carried out for 24 h at 37 °C according to a protocol of Williams et al. [29]. MS measurements were performed at an Ultraflex MALDITOF mass spectrometer (Bruker Daltonics, Bremen, Germany) applying following criteria: nitrogen laser ionization at

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337 nm, 50 Hz repetition rate, positive reflector mode at accelerating potential of 25 kV, delayed extraction, output signal digitalization at 1 GHz. Bruker Daltonics software “flex control (v2.0)” was used for measurements and “flex Analysis (v2.0)” for peak labelling. Resulting PMFs were used for protein identification by analysis with MASCOT software (Matric Sciences; parameters: enzyme: trypsin; modifications: Carbamydomethyl (C); missed cleavages: 0–1; peptide tolerance: ± 50 ppm; mass values: MH+ and monoisotopic; only protein scores (P) as represented by the probability-based Mowse score were considered for significant protein identification when scores were greater than 67) and the DFCI Medicago Gene Index v9.0 (at Dana-Farber Cancer Institute, Harvard School of Public Health, Boston, U.S.A.). In case of successful sequence matching in MtGI, sequences were verified by performing BlastP protein annotation of the in silico translation products at the US National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov/BLAST). Additional blast analyses of all sequences were carried out using the “Aphano DB” EST database (http://www.polebio.scsv.ups-tlse.fr/aphano/) and the S. meliloti genome project database (http://iant.toulouse. inra.fr/bacteria/annotation/cgi/rhime.cgi). Theoretical peptide mass and pI of the polypeptides were evaluated at EXPASy (http://www.expasy.org/tools/pi_tool) for final confirmation according to their positions in the 2-DE gel map. Prediction of signal peptide sequences for selected proteins was performed using the SignalP 3.0 server (http://www.cbs.dtu.dk/services/ SignalP-3.0/).

3.

Results and discussion

3.1.

Experimental setting

To monitor differentially induced protein patterns in the root proteome of the model legume M. truncatula after infections with symbiotic and pathogenic soil microorganisms at early stages of infection, an inoculation assay was established for M. truncatula (‘Jemalong A17’) plants growing on solid Fahraeus medium Petri dishes. Inoculation of plant roots was achieved for symbiotic interactions with the mycorrhiza fungus G. intraradices and the rhizobial bacterium S. meliloti 1021, and for the parasitic interaction with the oomycete A. euteiches. Roots of 10 days old seedlings were carefully inoculated with spores' inoculums in case of G. intraradices and A. euteiches infections or with a liquid bacterial culture for S. meliloti infection. Control roots were Mock inoculated with an equal volume of sterilized water.

Fig. 1 – A + B. Proteome maps from the 2D-DIGE analyses for M. truncatula A17 roots after Mock-inoculation (controls) and inoculation with the symbionts G. intraradices and S. meliloti as well as the pathogen A. euteiches, 6 h post inoculation (6 h). A) 2-D gel images of controls (con, labelled with Cy2), mycorrhized roots (myc, labelled with Cy3), rhizobia nodulated roots (nod, labelled with Cy5) and superimposition-image of all three fluorescence channels according to con-, myc- and nod-treatments. Differentially induced proteins that were identified by MS are numbered. B) 2-D gel images of A. euteiches-infected roots (Ae, labelled with Cy2), mycorrhized and A. euteiches-infected roots (myc + Ae, labelled with Cy3), rhizobia nodulated and A. euteiches-infected roots (nod + Ae, labelled with Cy5) and superimposition-image of all three fluorescence channels according to Ae-, myc + Ae- and nod + Ae-treatments. Differentially induced proteins that were identified by MS are numbered.

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Fig. 3 – The M. truncatula root “interactome” indicating all proteins found induced after G. intraradices, S. meliloti and A. euteiches infection in the root proteome of the legume after 6 and 24 h post infections. Proteins that were found induced during at least one of the interactions are numbered.

Separate infections with the three mentioned microorganisms applied on discrete M. truncatula plates were compared to mixed infections combining one primary symbiotic infection (whether G. intraradices or S. meliloti) with a secondary A. euteiches infection event at 3 h after primary infection. Plants were harvested at 6 h and 24 h of infection.

3.2.

2D-DIGE analysis

For identification of protein patterns induced in the M. truncatula root proteome by infections with the above mentioned microorganisms, total proteins were extracted from inoculated roots and Mock-inoculated control roots at the time-points of harvest 6 h and 24 h after infection. For differential two-dimensional gel electrophoresis (2D-DIGE), protein extracts were labelled with fluorescent dyes and combined prior to 2-DE separation following two experimental settings:

(a) For identification of protein patterns induced after symbiotic infections, protein samples of control roots (Cy2), G. intraradices infected roots (Cy3) and S. meliloti infected roots (Cy5) were combined. (b) To identify alterations among protein induction patterns according to mixed symbiotic and pathogenic infections, protein extracts of A. euteiches-infected roots (Cy2), G. intraradices and secondary A. euteiches-infected roots (Cy3), and S. meliloti and secondary A. euteichesinfected roots (Cy5) were combined. The three combined proteomes of each experimental approach (a, b) were separated on one gel but visualized differentially using a fluorescence imager resolving three different gel images according to the appropriate excitation wavelengths for the applied Cy dyes. However, the most evident differences among protein profiles for the two timepoints were immediately detectable from superimposition of

Fig. 2 – A + B. Proteome maps from the 2D-DIGE analyses for M. truncatula A17 roots after Mock-inoculation (controls) and inoculation with the symbionts G. intraradices and S. meliloti as well as the pathogen A. euteiches, 24 h post inoculation (24 h). A) 2-D gel images of controls (con, labelled with Cy2), mycorrhized roots (myc, labelled with Cy3), rhizobia nodulated roots (nod, labelled with Cy5) and superimposition-image of all three fluorescence channels according to con-, myc- and nod-treatments. Differentially induced proteins that were identified by MS are numbered. B) 2-D gel images of A. euteiches-infected roots (Ae, labelled with Cy2), mycorrhized and A. euteiches-infected roots (myc + Ae, labelled with Cy3), rhizobia nodulated and A. euteiches-infected roots (nod + Ae, labelled with Cy5) and superimposition-image of all three fluorescence channels according to Ae-, myc + Ae- and nod + Ae-treatments. Differentially induced proteins that were identified by MS are numbered.

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the 2-DE images of differentially treated and labelled samples (Fig. 1 (6 hpi), Fig. 2 (24 hpi): colored gels displayed below). Evaluation of relative in-gel protein abundances of differentially induced protein spots detected by using the DeCyder software allowed identification of 87 spots with at least 1.5fold changes in volume among all biological and technical repetitions carried out. Selection criteria were consistently detectable increases in protein abundances as compared to the corresponding abundances in control gels during the whole experiment. While 57 proteins were already induced at 6 hpi, a marked increase in protein abundance was observed at 24 hpi: 85 out of 87 induced proteins were detected. The early 6 hpi time-point is at the detection limit for identification of interaction-specific protein induction, and it has to be thoroughly distinguished from normal variations in cellular protein biosynthesis. Since this set of 87 proteins was found induced specifically after symbiotic and pathogenic infection including fungal as well as bacterial interaction partners, this protein profile may represent the early (24 h post infection) M. truncatula root “interactome” (Fig. 3). Thus, based on the detection of approximately 1000 different resolved protein spots per single gel, ∼ 9% of proteins are altered in abundance due to interactions with microorganisms (at 24 hpi). For identification of infection-specific protein induction, the above mentioned selection criteria were applied for evaluation of relative abundances between samples of different inoculation experiments. In detail, 31 proteins were detected to be solely induced after A. euteiches infection, plus 21 proteins with predominant induction after A. euteiches infection but also appearing during symbiotic or mixed infections (Fig. 1, Table 1). 11 proteins were found induced specifically during symbiotic infections, while additional 23 proteins are also apparent during mixed infections but exhibit predominance for symbiotic infections (Fig. 2, Table 2). Only one protein was found solely induced in gels of control tissue at 6 hpi (Table 2). For protein identification, selected spots were excised in duplicates from two different Coomassie-stained gels, digested with trypsin, and analysed by MALDI-TOF mass spectrometry. Subsequent alignment of the resulting PMFs with the DFCI Medicago truncatula Gene Index (MtGI) database led to identification of all 87 picked protein spots. By contrast, additional blast analyses of all sequences available in the “Aphano DB” EST database and the S. meliloti genome project database resulted in no matches found for any sequence analysed (for G. intraradices, currently no gene sequence information are accessible in public databases). Hence, all proteins identified were regarded as being of plant origin. The proteins are listed in Tables 1 and 2 according to their specific induction during pathogenic or symbiotic infection. Proteins are listed beginning with the proteins of highest relative in-gel intensities induced at both time-points in comparison to the correspondent proteins of other inoculation experiments, followed by those with steadily decreasing intensities or induction at the later 24 hour-time-point only and those that were found induced also during mixed infections. Data of standard deviation (SD) for one protein spot is displayed in brackets under abundance index only in case that the value exceeds a SD of ±0.5 (Tables 1 and 2). Detailed data of protein induction, SDs as well as calculated MW and pI values are

additionally displayed in the supplemental Table S1. The list of proteins allowed the determination of induced M. truncatula pathways during early interaction stages as noted in the following.

3.3.

Pathogen (A. euteiches)-induced protein patterns

18 proteins were found specifically induced during A. euteiches infection at both time-points (Table 1, upper section). This set of proteins comprises pathogenesis-related (PR-) proteins of class 10 (spots 11, 10, and 12) exhibiting the highest induction levels, followed by two osmotin-/thaumatin-like (PR5b) proteins (spots 24, and 89). Another PR10-like ABA-responsive protein (spot 13) was found induced at 24 hpi but not at 6 hpi. PR10 and PR5b protein induction was reported previously at both transcript [30] as well as protein level [24,25] to constitute the major molecular defense response of M. truncatula following A. euteiches infection mainly during advanced infection stages. In the present study, we could detect their pathogen-specific induction already at an early infection stage (6 hpi). Both PR protein classes were found to be conjointly modified in induction due to A. euteiches infections in M. truncatula [26,27]. Although they are structurally not related to each other, PR10s and TLPs share some comparable characteristics: both were proven for in vitro antifungal and also antioomycete activity and are specifically inducible after pathogenic challenge [31–34]. However, their exact biological role remains unclear. Structural analyses revealed pore- or cave-forming conformations for both protein classes suggesting a more general function in ligand-binding signalling or phytohormone homeostasis under stress conditions, especially for hydrophobic substrates such as (membrane) lipids, steroid-like compounds such as brassinosteroids (BRs) and – in case of PR10s – cytokinins and abscisic acid (ABA) [35–38]. As both PR protein classes were found specifically induced during early A. euteiches infection stages but neither after inoculation with the two microsymbionts nor during mixed infections, it is very likely that these proteins represent key players during initial pathogen defense-related signalling events distinct from symbiotic signalling pathways. Pathogen-specific induction was also noted for two Kunitztype proteinase inhibitors (ST1-like) (spots 19 and 88) and for a stress-induced ST1-like protein (spot 76). Proteinase inhibitors (PIs) are one of the most important classes of defense proteins in plants representing also one major defense–counterdefense mechanism occurring in plant-oomycete interactions [39–41]. When secreted into the medium, PIs are capable to suppress the growth of pathogens [42,43]. A ST1-like kunitz inhibitor was found secreted from M. truncatula 2HA cell cultures [44]. However, gene silencing of the pathogen-inducible M. truncatula (Kunitz-type) protease inhibitor MtTi2 did not directly influence M. truncatula root infection by A. euteiches, but led to transcriptional suppression of defense-associated genes including co-induced proteases and PIs, germin-like proteins, (glutathione) peroxidases, class 1 chitinases, flavanoid (UDP) glycosyltransferases, a lectin and a lipid transfer-like protein [45]. Here, a defense-related protein set including members of those mentioned was found induced together with Kunitztype PIs. Thus, Kunitz-type PIs seem to be capable of inducing defense-associated signalling.

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Protein 16 is a galactose-binding lectin induced 6 and 24 h after A. euteiches infection. Plant lectins represent a large group of carbohydrate binding proteins with diverse functional properties. Vegetative lectins were found to be specifically expressed in developing seedlings [46], while root lectins, especially those that possess signal peptides for the entry to secretory pathways, may be involved in interactions between plants and microorganisms. They may participate in recognition processes via carbohydrates exposed to the cell wall surfaces of microorganisms [47,48]. SignalP v3.0 predicts the existence of a signal peptide sequence of the N-terminal 26 aminoacids (probability: 0.771) for the lectin sequence described here. Hence, since this lectin was found specifically induced after A. euteiches infection with increased induction for the early 6 hpi time-point, it might be involved in pathogen recognition. Proteins 23, 62, 85, 59, 67, 86, 36, 50, 61, and 69 are related to primary carbohydrate and energy-related metabolism. Their induction is detected especially at 24 hpi and is supposed to be connected to an advanced infection process (see paragraph below).

3.4.

Predominantly A. euteiches-induced proteins

The protein profiles induced during mixed infections with microsymbionts (but with predominant A. euteiches-mediated induction) (Table 1, lower section) comprise primarily proteins involved in antioxidant defense and detoxification of ROS. An early plant response towards infections with (mainly pathogenic) microorganisms is the generation of ROS, but on the other hand also several antioxidant enzymes acting as interceptors are produced to protect the cell from oxidative damage. Induction of a cysteine synthase (spot 55) already at 6 hpi indicates activation of the glutathione-S-transferase (GST) redox system involved in primary antioxidation processes, where the availability of cysteine as sulphur amino acid precursor represents the major determinant. Other important enzymes of the antioxidant defense were found by detecting three peroxidases (spots 51, 71, and 72) and by the key enzyme superoxide dismutase (spot 4). Beside the notable accumulation of antioxidants, protein classes involved in defense mechanisms, which were already found induced during sole pathogenic infection, show enhanced induction during mixed infections. These induction patterns seem to reinforce the plants defense potential due to microsymbiotic co-infection. Moreover, significant decreases of PR5- and PR10-type proteins that were predominantly induced during A. euteiches infection indicate a bioprotective effect after symbiotic co-infection. Prominent protein patterns seen in advanced infection stages include proteins assignable to carbohydrate metabolism and energy-related processes (spots 58, 60, 63, 70, 75, 87, and 8), proteins associated with secondary phenylpropanoid as well as phytoalexin metabolism (spots 40, 46, 45, and 17) and proteins of the proteasome degradation system (spots 26, 39 and related: spot 1). Especially the conjoined induction of several proteins from the carbohydrate metabolism together with proteins from the phenylpropanoid metabolism is conceived to represent an effective defense response towards pathogenic infection [27]. Here, identification of an isoflavone reductase (IFR) and an

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IFR-like NAD(P)H-dependent oxidoreductase, a chalcone reductase, and a chalcone-flavone isomerase 1 indicate induction of the phenylpropanoid metabolism which is important for synthesis of M. truncatula pterocarpan phytoalexin medicarpin, the major phytoalexin in Medicago ssp. [49,50]. Interestingly, beside the predominant A. euteiches-related induction, this proteins pattern was also observed during G. intraradices inoculation and during mixed G. intraradices/A. euteiches inoculations (NAD(P)H-dependent 6′-deoxychalcone synthase (spot 43), Table 2). Coincidently, an accumulation of key enzymes from the flavanoid pathway leading to medicarpin biosynthesis was reported for M. truncatula roots during mycorrhizal association, putatively mediating a bioprotective effect towards pathogenic colonization by triggering the molecular plant defense [19,25]. Hence, induction of phenylpropanoid responses is supposed to occur at very early infection stages (6 hpi).

3.5. Symbionts (G. intraradices, S. meliloti)-induced protein patterns 11 proteins were detected with specific induction after inoculation either with G. intraradices and/or S. meliloti (Table 2, upper section). Among this induction pattern, calmodulin-2 (spot 9) represents the protein with the highest abundance after infection with both symbiotic interaction partners. Calmodulin (CaM) is a calcium-binding regulatory protein ubiquitously present in all eukaryotes which encompasses a broad range of cellular functions. Calcium is a common secondary messenger, and calcium oscillations (“spiking”) have the capacity to transduce information from the perception of a ligand signal or environmental change, leading to activation of downstream responses [4]. This calcium signalling also plays a central role in early pathways of symbiotic associations both with rhizobial bacteria and arbuscular mycorrhizal fungi, where perception of microbial (nod-; myc-) signals is activating a cascade that converges on the nucleus including transcriptional reprogramming [5]. Among that, the DMI3 gene encodes a calcium- and calmodulin-dependent protein kinase (CCaMK) that functions immediately downstream of nuclear calcium spiking. CCaMK could bind calcium directly via EF-hand domains or indirectly as a complex with calmodulin [51]. As CaM-2 is strongly induced after symbiotic infections, it might be a part of early myc- and nod-signalling pathway. In accordance with an enhanced nucleocytoplasmic signal transduction, two isoforms of a Ran-binding protein were found induced, one after nodulation (spot 30) and the other following mycorrhization (spot 38). Here, the nuclear membrane regulates nucleocytoplasmic shuttling of proteins and RNA thereby controlling signal transduction and downstream gene expression [52]. Delivery through the nuclear pore complex (NPC) is achieved by importins (α, ß), together with ras-related small G-proteins, RanGDP and RanGTP-nucleoporins and its binding proteins (RANBPs). This affects also regulatory proteins involved in plant hormone homeostasis and signalling [53]. Likely, during mixed but predominantly symbiotic infections, a protein of auxin signalling (spot42, auxin-induced protein) was identified (Table 2, lower section). Noticeably, many proteins involved in energy-regulatory processes are significantly induced due to both symbiotic

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associations. This set includes an ATP synthase subunit d (spot 21), a nucleoside diphosphate kinase (spot 7) involved in ADP synthesis, and two glycolytic key enzymes, the malate dehydrogenase (spot 47) and a triosephosphate isomerase (spot 27), beside several enzymes of primary metabolism found during mixed infections but with predominant induction during symbiosis (see next paragraph). A putative adenosine kinase 2 (spot 54) involved in purine synthesis was also found induced. Much likely, these energetic molecules are required for phosphorylation/dephosphorylationdependent early signalling events as described above. The induction of the two glycolytic enzymes in mycorrhized roots indicates that increased carbon fixation is initiated for supplementation during the mutualistic plant–fungi interaction. With the acid phosphatase (ACP, spot 28), a putatively nodulation-specific protein was found induced, coincidently only in S. meliloti infected roots. This protein was identified as the major ACP from soybean during nodulation [54]. There, it was found to be specifically involved during symbiosis-related nitrogen fixation in form of the ureides allantoin and allantoic acid, which are derived from de novo-synthesized purines. The first step of purine conversion into ureide is the removal of the 5′-phosphate group that is assumed to be achieved specifically by this ACP form, since its enzymatic activity was found dramatically higher than that of the presumed rate-limiting enzyme in ureide synthesis, the phosphoribosylpyrophosphate aminotransferase [54]. Nitrogen assimilation by the plant is supposed to be in preparation, since a P5C dehydrogenase (spot 79) was found induced in nodulated roots, that is involved in degradation of proline into glutamate.

3.6. Predominantly symbiosis (G. intraradices, S. meliloti) -related protein induction Coincident with the protein profiles induced during mixed infections but with predominant A. euteiches-mediated induction (Table 1, lower section), the protein pattern induced during mixed infections but with predominance in symbiotic associations (Table 2, lower section) comprises several proteins involved in antioxidant defense. This protein set includes a stromal ascorbate peroxidase (spot 44), a peroxidase (spot 22) and a cationic peroxidase 1 (spot 74), and a patatin-like protein (spot 64) involved in HR reactions. Furthermore, identification of a cytochrome c oxidase subunit (spot 52) together with the mitochondrial processing peptidase belonging to cyt c reductase (spot 44) as well as two aldehyde dehydrogenases (spots 84, and 83) indicates an initiated mitochondrial antioxidative response. The observation of ROS-intercepting enzymes induced under predominant symbiotic influence underlines the importance to control ROS accumulation also during symbiosis. Induction of oxidative stress protectors together with that of signalling factors and enzymes of energy-regulatory pathways (as shown by the symbiotic protein profiles, Table 2, upper section) probably represents the prerequisite for beneficial effects on growth as well as increased pathogen tolerance. While the protein induction patterns during mixed infections but with predominant A. euteiches-mediated induction comprise mainly defense-associated functional clusters of A.

euteiches-induced profiles, the protein patterns under predominant symbiotic induction reveal mainly signalling elements and enzymes involved in growth regulation. Interestingly, induction of PR proteins remains absent, indicating a reduced defense response during co-infection with microsymbionts. Two ß-glucosades G1 were detected, one with high induction levels (spot 78) during myc- and myc/Ae-infections, and another (spot 82) weakly induced in nod- and myc/Ae-/nod/ Ae-infections. These enzymes are involved in cellulose degradation and their induction indicates comprehensive restructuring at the root surface due to penetration of microorganisms. Investigations suggest the existence of very similar or at least partly overlapping mechanisms in penetration responses to pathogenic fungi and oomycetes with AM [10]. These mechanisms suggest the initiation of localized secretory activity [10], which might be enhanced during mixed infections of a fungal and fungus-like effector. With protein no. 65, an ankyrin-repeat protein was found induced. Such proteins have been described as interacting partners of ß-glucanases implicated in pathogen defense, but a clear functional characterization is still elusive [55]. Very recently, ankyrin-repeat proteins were found to enhance auxin-mediated transcription accomplished by nuclear bZIP transcription factors [56]. It was found that increased cellular auxin concentrations result in the accumulation of originally cytosolic ankyrin-repeat proteins in the nucleus. Interestingly, with detection of an auxin-induced protein (spot 42), auxinrelated signalling is supposed to be induced during both mycorrhization and nodulation as well as mixed infections. When auxin stimulates the cell, auxin repressor-proteins are degraded by the ubiquitin-dependent proteolytic pathway (note the induction of a proteasome subunit (spot 48) also during mixed infections with symbiotic predominance). On the other hand, several regulatory factors involved in auxin signalling are transported into the nucleus [53]. Interestingly, the already described Ran-binding proteins (RanBPs) found induced during symbiotic associations (spots 30, and 38) are supposed to play a fundamental role in auxin signalling, since auxin sensitivity was drastically altered in RanBP transgenic plants [57]. Thus, antisense transgenic plants revealed longer primary roots but retarded lateral root growth. Putatively, stimulation of root development during interactions with symbiotic microorganisms is achieved via induction of auxin-mediated pathways. Auxin is supposed to play a major role in lateral root formation and elevated endogenous auxin levels significantly increased root branching [58,59]. A similar effect was also observed at different stages in root nodule organogenesis during rhizobial symbiosis [60]. Moreover, proteomic studies in M. truncatula revealed an >80%-overlap of protein changes during early responses (24 hpi) to S. meliloti with responses to auxin, indicating consistent molecular mechanisms and the importance of auxin signalling during initial nodulation stages [61,62]. The role of auxin during AM symbiosis is much less investigated. Recently, Campanella et al. [63] could show increased levels of the auxin indole-3-butyric acid (IBA) in G. intraradicesinoculated M. truncatula roots. Since it was indicated that lateral root formation is stimulated during AM symbiosis via the “DMI1/DMI2 signalling pathway” [7], activation of auxin synthesis may represent the executing mechanism downstream of the initial signalling pathway.

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The overall dominant proteins induced during mixed but predominant symbiotic infections are 14-3-3-like proteins that were found induced with 4 isoforms (spots 25, 31–33) representing two different proteins. 14-3-3 proteins belong to a conserved kinase-like protein family that are known to regulate different cellular processes, such as apoptosis, signalling, carbon and nitrogen metabolism [64]. Their targets are proteins and most 14-3-3-target proteins identified to date in plants are metabolism-related enzymes. Thus, 5 of the 10 glycolytic enzymes and 7 of the 13 enzymes participating in the Calvin cycle were found to be 14-3-3-interactors [64]. Here, in coincidence with a predominant 14-3-3 protein induction in mycorrhized roots, glycolytic enzymes were found significantly induced after AM infection (spots 47 and 27) or predominantly myc-induced (spot 66), indicating increased carbon metabolism during AM.

3.7.

Summary and conclusions

We have applied a two-dimensional differential gel electrophoresis approach to identify protein patterns in the M. truncatula root proteome that are specifically induced during early association events with symbiotic as well as pathogenic soil microorganisms and during mixed infections at 6 and 24 hpi. We are confident that our 2D-DIGE analyses led to the identification of interaction-specific protein profiles whose qualitative evaluation is supposed to contribute to a more profound understanding of early molecular events during plant–microbe interactions at the proteome level. Thus, a set of 87 proteins likely represents the M. truncatula root “interactome” during early infection time-points, covering fungal, oomycete as well as bacterial key interactors of legumes. As the mechanisms of symbiotic and pathogenic organisms for ingress into the host plant resemble each other, the primary molecular defense reaction during pathogenic, symbiotic or mixed infections is very similar. It comprises proteins involved in antioxidant defense and/or attack with reactive oxygen species. Symbiotic AM fungi seem to have evolved mechanisms to circumvent the initial host defense, which may explain the broad host range of AM fungi. One putative strategy is to limit the contact with the host defense system, which already indicated the special organization of the mutualistic interaction, starting with the development of an AM prepenetration apparatus, intracellular hyphae growth and arbuscule formation. Moreover, suppression of host defense was observed in arbuscules and adjacent cells but not in non-colonized regions of the host root [65]. After the primary defense reaction, pathogen and symbiosis-induced protein profiles in M. truncatula appear to be distinct from each other, indicating the induction of also very different molecular pathways. While A. euteiches induced a set of defense-related proteins (that was absent during symbiotic infections), consisting of mainly PR proteins, Kunitz-type proteinase inhibitors, a lectin, and proteins related to primary carbohydrate metabolism, symbiosis-related protein induction includes a typical factor of early symbiosis-specific signalling (CaM-2), two RanBPs of nucleocytoplasmic signalling, and coincidently a set of energy-related enzymes together with specific proteins that gave hints to symbiosisinitiated C- and N-fixation. Latter mentioned symbiosis-

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mediated protein pattern is assumed to represent the initial or basic prerequisite for beneficial or even protective effects for the plants. Generally, differential protein induction due to early symbiotic infections was detected in lesser quantity as compared to that during early pathogenic infection. Reasons therefore are likely prolonged symbiotic infection and preinfection periods. For the rhizobial interaction, minimal infection time is assumed to represent > 24 hpi and nod genes regulation becomes significant from 12 hpi on [66,67]. For the mycorrhiza interaction, establishment of novel intracellular structures in the plant epidermis cells is assumed to require 4 to 5 h after initial contact with hyphae was achieved, and within this time-period nod genes activation is initiated [11]. With respect to hormone-regulation, A. euteiches pathogenic infection led to induction of ABA-related pathways, while there is indication that auxin-related pathways are induced during symbiosis. Induction of enzymes from the phytoalexin synthesis was observed mainly for A. euteiches infections, but also during AM interaction. The protein induction during mixed infections is primarily of interest with respect to molecular indications for putative beneficial effects for the plant by symbionts and even “bioprotective” effects towards pathogenic infections resulting from co-infections with symbionts. Thus, specific pathogenesis-mediated protein induction is supposed to appear decreased during symbiotic co-infections as compared to single pathogenic infections. This is exactly the case for the PR5- and PR10-type proteins that were found predominantly induced during A. euteiches infection: they are absent, either during mixed infections, but also during solely symbiotic infections. Since their induction is supposed to be directly correlated to pathogenic infections, a reductive effect due to the presence of microsymbionts becomes likely. On the other hand, the data for mixed infections allow a qualitative gradation of the protein induction patterns concerning the predominant influence of whether pathogenic or symbiotic inoculations. While the protein profiles with predominant pathogenic induction show defense-associated patterns that resemble those already found induced after sole A. euteiches infection, protein profiles with predominant symbiotic induction consist mainly of signalling elements and enzymes involved in hormonal (auxin) regulation. This phenomenon might be a result of a retarded establishment of symbiotic infections as compared to pathogenic infection events, or might represent an improvement of symbiotic signalling due to the pathogenic challenges. Interestingly, a set of 14-3-3-like proteins was significantly induced during mixed infections but predominantly during symbiotic interactions. Since the list of putative 14-3-3 interactors is extensive, but probably with preferential origins from signalling pathways and carbon and nitrogen metabolism, these proteins could represent the connecting elements between symbiosis- and pathogenesis-mediated early infection responses. Assumedly, an early and expanded induction of cellular factors involved in diverse signalling processes together with a set of antioxidants as it was observed for symbiotic infections, could pre-trigger the plant′s defense potential towards increased tolerance during pathogen infection. To our knowledge, comparative proteomic analyses of early plant responses to symbiotic, pathogenic and mixed

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infections are described here for the first time. However, further in-depth analyses for elucidation of downstream components of this signalling and identification of their major targets have to be carried out.

Acknowledgments The authors would like to thank Mrs. Nadine Küpper, University of Bielefeld, Dept. 7, Proteome and Metabolome research, for her assistance in performing the MALDI-TOF-MS and database analysis. We further are grateful to Prof. Dr. Helge Küster, Leibniz University of Hannover, Institute for Plant Genetics, Dept. IV, Plant Genomics, for the helpful discussions. The Deutsche Forschungsgemeinschaft (DFG) supported this work.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jprot.2009.10.009.

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