rsc mb c3mb70069k 3.a department of systems biology, agricultural biotechnology research institute...

This journal is c The Royal Society of Chemistry 2013 Mol. BioSyst.

Cite this: DOI: 10.1039/c3mb70069k

A proteomics approach to study the molecular basis ofenhanced salt tolerance in barley (Hordeum vulgare L.)conferred by the root mutualistic fungusPiriformospora indica†

Mehdi Alikhani,zab Behnam Khatabi,zc Mozhgan Sepehri,d

Mojtaba Khayam Nekouei,e Mohsen Mardie and Ghasem Hosseini Salekdeh*abe

Piriformospora indica is a root-interacting mutualistic fungus capable of enhancing plant growth,

increasing plant resistance to a wide variety of pathogens, and improving plant stress tolerance under

extreme environmental conditions. Understanding the molecular mechanisms by which P. indica can

improve plant tolerance to stresses will pave the way to identifying the major mechanisms underlying

plant adaptability to environmental stresses. We conducted greenhouse experiments at three different

salt levels (0, 100 and 300 mM NaCl) on barley (Hordeum vulgare L.) cultivar ‘‘Pallas’’ inoculated with

P. indica. Based on the analysis of variance, P. indica had a significant impact on the barley growth and

shoot biomass under normal and salt stress conditions. P. indica modulated ion accumulation in

colonized plants by increasing the foliar potassium (K+)/sodium (Na+) ratio, as it is considered a reliable

indicator of salt stress tolerance. P. indica induced calcium (Ca2+) accumulation and likely influenced the

stress signal transduction. Subsequently, proteomic analysis of the barley leaf sheath using two-

dimensional electrophoresis resulted in detection of 968 protein spots. Of these detected spots, the

abundance of 72 protein spots changed significantly in response to salt treatment and P. indica-root

colonization. Mass spectrometry analysis of responsive proteins led to the identification of 51 proteins.

These proteins belonged to different functional categories including photosynthesis, cell antioxidant

defense, protein translation and degradation, energy production, signal transduction and cell wall

arrangement. Our results showed that P. indica induced a systemic response to salt stress by altering the

physiological and proteome responses of the plant host.

Introduction

Abiotic stresses, including drought and salinity, are the majorlimiting factors of crop production worldwide. To improve

growth performance and evade stresses, one strategy for a plantis to establish associations with the beneficial microbial micro-organisms.1 Mutualistic symbiosis with mycorrhizal and endo-phytic fungi can enhance plant stress tolerance by increasingthe fitness of their host.2 The most ancient mutualisticsymbionts are arbuscular mycorrhizal fungi (AMF).3 Endo-phytes are another group of symbionts that accomplish partof their life cycle within the living host tissues without causingapparent damage to hosts.4 Endophytes may provide protectionand improve survival of their hosts, thereby resulting in anenhanced resistance to stress, insects, and disease as well asimproving the host yield.4

Piriformospora indica is a root endophytic fungus, first iso-lated from the Indian Thar desert and recognized as a plantroot symbiont.5,6 P. indica is a member of Sebacinales and so farall members of this order are involved in mycorrhizal associa-tions encompassing ecto-, orchid-, ericoid-, cavendishioid- and

a Department of Systems Biology, Agricultural Biotechnology Research Institute of

Iran, P.O. Box 31535-1897, Karaj, Iran. E-mail: [email protected];

Fax: +98-26-32704539; Tel: +98-26-32703536b Department of Molecular Systems Biology, Cell Science Research Center,

Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iranc Department of Entomology and Plant Pathology, University of Tennessee,

Knoxville, Tennessee, USAd Department of Soil Science, Faculty of Agriculture, Isfahan University of

Technology, Isfahan, Irane Department of Genomics, Agricultural Biotechnology Research Institute of Iran,

Karaj, Iran

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3mb70069k‡ These authors contributed equally.

Received 18th February 2013,Accepted 8th March 2013

DOI: 10.1039/c3mb70069k

www.rsc.org/molecularbiosystems

MolecularBioSystems

PAPER

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Mol. BioSyst. This journal is c The Royal Society of Chemistry 2013

jungermannioid mycorrhizas with plants.7 Since AMF cannotbe grown outside of their host plants, a problem is encounteredboth in commercial usage and in the study of the underlyingmechanism of AMF. P. indica, however, can be grown in theaxenic culture and can improve the plant growth rate in a widerange of plants.8,9 Nevertheless, the detailed molecular mecha-nism by which P. indica induces plant growth promotion is stillnot very clear. P. indica provides several more benefits to thehosts other than merely plant growth promotion, such as ahigher tolerance to various biotic and abiotic stresses.9 In addi-tion, it improves plant fitness by increasing the plant growthperformance both under normal and stressful conditions.8,10–12

It is likely that the mechanisms leading to abiotic stress (such assalt and drought) conferred by P. indica are based on the general,and not specific, plant-species mechanisms.12

Salinity is one of the major abiotic stresses that restrictsplant growth, development and productivity.13 Particular atten-tion has been paid to the effects of salt stress on the morpho-logical, physiological and cellular aspects of the host plants.However, the molecular mechanism underlying the salt tolerancehas been less studied. The contribution of P. indica to improvesalt tolerance in barley has been explored in several studies.8

P. indica has been found to elevate the plant antioxidantcontent to minimize the effect of salinity on barley.14 However,the detailed molecular mechanism associated with salt stresstolerance conferred by P. indica in barley is unclear.

Proteomics has proven to be a powerful approach in studyingplant response to biotic and abiotic stresses.15–17 The applicationof proteomics can shed light on the mechanisms behindP. indica-mediated salt tolerance. Analyzing the changes inphysiological traits and protein expression reveals some aspectsof the adaptive mechanism occurring in P. indica-colonizedplants. Presently, only a few proteomic studies are investi-gating the adaptive mechanisms of plant symbionts to theenvironmental stresses which are particularly important foragriculture.18,19 However, to the best of our knowledge, this isthe first report showing the proteome analysis of symbiontsunder saline conditions.

We analyzed the proteome patterns of barley leaf sheaths inP. indica-colonized plants and then compared the colonizedplants with control plants under different salt stress conditionsusing a two-dimensional gel electrophoresis (2-DE) basedproteomics approach. We then coupled physiological data withdata collected from mass spectrometry and identified differen-tially accumulated proteins associated with salinity andP. indica-root colonization. Our results showed that P. indicareprogrammed the host physiology by altering the ion content,and proteome patterns of barley leaf sheaths in order to copewith salt stress.

Materials and methodsPlant materials, inoculation, growth conditions, salt treatmentand experimental conditions

Barley seeds of the cultivar ‘‘Pallas’’ were surface-sterilizedwith 70% ethanol (v/v) for 30 seconds followed by 6% sodium

hypochlorite (NaOCl) for 15 minutes. They were then rinsedthoroughly with sterile water, and germinated for 2 days.P. indica was cultured on CM (complex medium) and the sporesuspension collected after 28 days by gently scratching thefungus surface on the petri dishes with a spatula until thespores were released. The spore suspension was filteredthrough cheesecloth to remove the excess medium and washedthree times with distilled water (dH2O) containing Tween-20.After each washing step, the spores were collected by centri-fugation at 4000g for 7 minutes. The spore pellet was finallysuspended again in dH2O and adjusted to B5� 105 spores per mL.The two-day-old barley seedlings were inoculated by immersingin the spore suspension solution with gentle shaking fortwo hours. The mock-treated seedlings were dipped in sterilewater only. Inoculated and mock-treated seedlings werelater transferred into pots, filled with sand and perlite in theratio of 2 : 1 and then placed in the greenhouse (16/8 h,day/night cycle, 22/18 1C regime and a 60% relative humidity).After fourteen days, the barley plants were exposed to 100 and300 mM NaCl. To eliminate the effect of osmotic stress shock,plants were treated gradually by increasing the applied saltconcentrations until the desired salt concentration was reachedafter seven days. The control group was grown under normalconditions (0 mM NaCl). The experiment was performed usinga completely randomized design with four independentbiological replicates. The leaf sheaths of the salt-treatedP. indica-colonized barley and non-inoculated plants wereharvested 14 days after salt treatment. Three plants per pot(as one replication) were harvested for the physiological andproteomic analysis.

Microscopic analysis

To check for the successful barley root colonization by P. indicaunder salt conditions, P. indica-inoculated barley roots wereharvested 14 days after inoculation. The root samples wereboiled in 10% KOH solution for 1 minute, washed three timeswith PBS (phosphate-buffered saline) and stained with 0.02%Trypan blue. In order to detect the fungi, root samples weredestained with 50% lacto-phenol and analyzed under a lightmicroscope.

Physiological analysis

After harvesting plant materials, the shoot and root freshweights were measured. The dry weight was then measuredafter drying fresh tissue in an oven at 70 1C for 48 hours.Physiological analyses were conducted by measuring the ionaccumulation including sodium (Na+), potassium (K+) andcalcium (Ca2+) after being extracted in 500 mM HNO3. Theion concentrations were measured in six replicates usinginductively coupled plasma optical emission spectrometry(ICP-OES) equipment and reported as mg g�1 dry weight.In addition, the foliar K+/Na+ ratio of P. indica-colonizedand non-inoculated barley plants was compared underboth the control and the different salt treatments (100 and300 mM NaCl).

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Protein extraction

Proteomic analysis of barley leaf sheath from P. indica-colonizedand non-inoculated plants was performed under both the con-trol and the different salt stress conditions. Protein extractionand 2-DE analyses were performed as previously described.20

Leaf sheath samples (1 g) were pulverized to a fine powder withliquid nitrogen using a mortar and pestle. Protein extractionwas performed via Trizol reagent (Invitrogen/Life technologies)according to manufacturer’s instructions. The leaf sheathpowder was suspended in 1 mL Trizol solution, homogenizedand kept at room temperature (RT) for 10 minutes. Two-hundred mL chloroform was added to the mixture and aftervortexing, the solution was held at RT for 5 minutes. Thesolution was then centrifuged at 12 000g for 15 minutes(all the centrifugation steps were conducted at 4 1C). After-wards, the supernatant containing the protein was transferredto a 2 mL fresh tube and acetone was added in the ratio of 3 : 1.Samples were vortexed vigorously and held at RT for 10 minutes.The protein was precipitated by centrifugation at 12 000g for10 minutes. Protein pellets were washed three times using 96%ethanol (containing 300 mM guanidine and 2.5% glycerol)followed by another wash with absolute ethanol containing2.5% glycerol. The protein pellets were then solubilized in lysisbuffer [7 M urea, 2 M thiourea, 2% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.8% (w/v)Pharmalyte pH 3–10, 1% (w/v), dithiothreitol (DTT)]. The pro-tein concentration was determined by Bradford assay (Bio-Rad)and Bovine Serum Albumin (BSA).

Two-dimensional electrophoresis and image analysis

Two-dimensional electrophoresis was performed as previouslydescribed.21 Immobilized pH gradient gels (IPGs) (24 cm,pH 4–7 IPG strips) were rehydrated overnight with 450 mL ofrehydration buffer [8 M urea, 0.5% CHAPS, 20 mM DTT, 0.5%(v/v) IPG buffers] in a re-swelling tray (GE Healthcare) at RT. Foranalytical and preparative gel electrophoresis studies, 300 mgand 3 mg of protein samples were loaded on IPG gels, respec-tively. Isoelectric focusing (IEF) was conducted using aMutiphor II and a DryStrip kit (GE Healthcare) at 20 1C. Thesamples were run at 300 V for 30 minutes, 500 V for 1 hour,followed by 1 hour at 1000 V and finally 3500 V for 16 hours.The focused strips were equilibrated twice for 15 minutes in10 mL equilibration solution. The first equilibration was per-formed in an equilibration solution [6 M urea, 30% (v/v)glycerol, 2% (w/v) SDS, 1% (w/v) DTT and 50 mM Tris-HClbuffer, pH 8.8], followed by a second equilibration in a similarsolution in which DTT was replaced with 2.5% (w/v) iodoacet-amide. Separation in the second dimension (SDS-PAGE) wasperformed in a vertical slab of acrylamide (12% total monomerwith 2.6% crosslinker) using a PROTEAN II Multi Cell(Bio-Rad). Protein spots in analytical and preparative gels werestained with silver nitrate22 and coomassie brilliant bluecolloidal (CBB) G-250, respectively.23 Gels were scanned usingGS-800 densitometer (Bio-Rad) at a resolution of 600 dots persquare inch. The scanned gels were analyzed using Melanie 4

software (GeneBio, Geneva, Switzerland). The molecular massesand pI (isoelectric point) of the protein spots on gels weredetermined by co-electrophoresis of standard protein markers(GE Healthcare) and migration of the protein spots on strips,respectively. Percent volume of each spot was estimatedand analyzed to determine protein abundance. A single two-dimensional gel per plant was run for each of four biologicallyindependent replicates and the percent volume of each spotwas estimated and analyzed. The two treatments (P. indica-colonized and non-inoculated) and three salt levels (0, 100 and300 mM NaCl) and four combinations were analyzed by two-wayanalyses of variance (ANOVA). ANOVA was performed by SPSSsoftware and mean comparisons were performed usingDuncan’s multiple range tests at a p-value r0.05, when appro-priate. Spots were determined to be significantly up- or down-regulated when the p-value is r0.05. The induction factor wascalculated by dividing the percent volume of spots in gelscorresponding to 100 and 300 mM NaCl by the percent volumeof spots corresponding to 0 mM NaCl.

Protein identification and database search

Protein spots were excised from CBB-stained gels and analyzedusing a Bruker Ultraflex III MALDI TOF/TOF mass spectrometeras described previously.24 Bruker flex analysis software wasused to perform the spectral processing and to generate thepeak lists for the MS and MS/MS spectra. The combined MSand MS/MS data were subjected to database searching using acopy of Mascot version 2.1 (Matrix Science Ltd.) that was runlocally through the Bruker BioTools interface (version 3.1). Thesearch criteria included enzyme, trypsin; variable modifica-tions, oxidation; peptide tolerance, 100 ppm; MS/MS tolerance,0.5 Da; instrument, MALDITOF/TOF. The search includedcarbamidomethyl as a fixed modification for all alkylatedsamples. The database search was run against NCBI non-redundant protein database NCBInr 20090222 (26 August2009: 7 894 593 sequences; 2 721 452 874 residues). The thres-hold for positive identification was a MOWSE score of >70(p-value r 0.05).

ResultsHost beneficiaries from P. indica under salt stress conditions

We first characterized the interaction of P. indica with barleyplants under different salt stress conditions. P. indica promotedthe barley growth under normal and salt stress conditions(Fig. 1) and that effect could be observed during the wholegrowth and development stages of barley plants. Overall,P. indica-colonized barley plants appeared larger, strongerand contained more leaves compared with non-inoculatedplants. Based on the microscopic analyses, P. indica colonizedbarley roots efficiently both under control and salt treatmentsas was seen by production of the number of chlamydospores at14 days after spore inoculation. No colonization was observedin non-inoculated plant roots. P. indica-root colonization wasbegun with the germination of chlamydospores that penetratedthe root cortex, ramifying intercellularly from the point of

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penetration and subsequently, formed chlamydospores withincortical cells (Fig. 2). After 4 weeks of barley development, thetotal fresh and dry shoot weights of P. indica-colonized plantswere increased up to 26% and 24%, respectively, as comparedwith non-inoculated plants under control conditions (0 mMNaCl) (Fig. 3). P. indica caused no visible changes in the rootmorphology. The effect of P. indica was expressed by thenumber of tillers and the leaf sheath expansion of colonizedbarley. Salinity negatively affected the barley growth in bothnon-inoculated and P. indica-colonized barley plants, as theshoot fresh and dry weights of colonized plants were signifi-cantly reduced by 43% and 26%, respectively, compared to51% and 40% reduction in the shoot fresh and dry weights of

non-inoculated plants, respectively (Fig. 3). The shoot freshand dry weights of P. indica-colonized plants were 1.53 and1.44 times higher, respectively, as compared to shoot fresh anddry weights of non-inoculated plants under severe salt condi-tions (300 mM NaCl) (Fig. 3).

Fig. 1 Comparison of plant growth phenotype of Piriformospora indica-colonized and non-inoculated barley cultivar ‘‘Pallas’’ under control (0 mM NaCl) and saltstress (300 mM NaCl). Plants were maintained in greenhouse (22 1C) until photographed at 28 days after inoculation.

Fig. 2 Piriformospora indica-colonized barley root at 14 days after inoculationunder salt treatment (300 mM NaCl). After spore germination, the funguspenetrates epidermal cells and spreads throughout the tissue and the sporesare formed both inter- and intracellularly. The barley root was stained withTrypan blue and visualized under a light microscope. The root endodermis andcentral cylinder is not colonized by the fungus. Intracellular spores are shown byarrows (bar = 30 mm).

Fig. 3 Effect of Piriformospora indica on shoot fresh weight, shoot dry weight,under different salt conditions (0, 100 and 300 mM NaCl). Graphs indicate asignificant difference between P. indica-colonized and non-inoculated plants atdifferent salt conditions. Bars indicate standard derivation values for eachtreatment.


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Physiological response of P. indica on barley plants undersaline conditions

The Na+, K+ and Ca2+ concentrations were measured in barleyleaf tissue of colonized and non-inoculated barley plants thatwere grown both under normal and salt conditions. Our resultsshowed that the Na+ concentration in leaves of both P. indica-colonized and non-inoculated barley plants were significantlyaffected by salinity (Table 1). The Na+ accumulation in theleaves of colonized and non-inoculated plants was increased upto 7 and 13 times, respectively, under severe salt conditions(300 mM NaCl). However, the K+ accumulation decreased by 7%and 2% in non-inoculated and colonized plants, while the Ca2+

concentration increased by 8% and 10% in non-inoculated andcolonized plants (Table 1). The distribution patterns of Na+ andK+ in barley roots were not significantly changed betweenP. indica-colonized and non-inoculated barley plants undernormal and salt conditions. Although, the foliar K+/Na+ ratioin P. indica-colonized plants was higher compared with non-inoculated plants under salt stress conditions (2.13 in P. indica-colonized plants compared with 1.91 in control plants undersevere salt conditions) (Table 1).

Identification of proteins in response to salinity andP. indica-root colonization

The protein patterns of barley leaf sheath of P. indica-colonizedbarley plants were compared with mock-treated plants underdifferent salt stress treatments (0, 100 and 300 mm NaCl).In total, 968 protein spots were detected in four independentbiological replicates (Fig. 4). Of these detected spots, 72 proteinspots showed significant differences in their abundancebetween P. indica-colonized plants compared with non-inoculated plants in response to salinity. Protein identificationvia Matrix-assisted laser desorption/ionization-Time Of Flight(MALDI-TOF/TOF) resulted in identification of 51 proteins(Table 2). Of the 51 identified protein spots, 18 protein spotsshowed changes in abundance in response to 100 mM NaCl inP. indica-colonized and/or non-inoculated plants under controlconditions. Of these, the abundance of 17 protein spots wasalso changed in response to 300 mM NaCl in P. indica-colonizedand/or non-inoculated plants under control conditions.The plant response to salt stress was more pronounced undersevere salt treatment (300 mM NaCl). We observed changesin the abundance of 50 protein spots under 300 mM NaCl inP. indica-colonized and/or non-inoculated plants under controlconditions.

Classification of altered proteins in response to 100 mM NaCland P. indica-root colonization

We found several proteins showing significant alteration in theexpression levels under 100 mM NaCl and these were cate-gorized into five groups (Fig. 5 and Table S1, ESI†). Thesegroups included group (A) with a single member, 6-4 photolyase(protein spot 817) which decreased in P. indica-inoculated andnon-inoculated plants both under 100 and 300 mM NaClconditions. The group (B) three proteins included nucleartransport factor 2 (protein spot 8), translation elongationfactor EF-Tu (protein spot 606) and Ribulose bisphosphatecarboxylase (Rubisco) small chain (protein spot 884) whichwere reduced in response to both 100 and 300 mM NaCl inthe non-inoculated barley plants. P. indica-root colonizationresulted in abundant recovery of all three proteins under mildsalt conditions (100 mM NaCl). However, the abundance of twoproteins (nuclear transport factor 2 and Rubisco small chain)returned to control levels in the P. indica-colonized plantsunder 300 mM NaCl. (C) ubiquinol-cytochrome c reductase(protein spot 928) was higher in all salt treated plants comparedwith the control condition except for inoculated plants under100 mM NaCl. The group (D) seven proteins including cyto-chrome c oxidase polypeptide Vb (protein spot 140), 23 kDajasmonate-induced protein (protein spot 301), tubulin-foldingcofactor A (protein spot 95), ribosomal protein P1 (proteinspot 117), cytochrome c oxidase subunit Vb (protein spot 122),peroxiredoxin-2E-2 (protein spot 119) and Rubisco small chain(protein spot 11) were up-regulated only in P. indica-colonizedplants. All these proteins were increased in P. indica-colonizedand non-inoculated plants at 300 mM NaCl. Finally, in-group(E) six proteins were found to increase at both levels of salinity.These proteins included vacuolar proton-translocating ATPase(V-ATPase) subunit E (YLP) (protein spot 473), Mg-chelatasesubunit (protein spot 621), Rubisco small chain (protein spot 66),endotransglucosylase/hydrolase XTH2 (protein spot 968),a-expansin EXPA2 (protein spot 205) and RNase S-like protein(protein spot 219). In this group, P. indica-root colonizationresulted in the recovery of only YLP in response to 300 mMNaCl treatment.

Classification of altered proteins in response to 300 mM NaCland P. indica-root colonization

Fifty protein spots were found to change abundance undersevere salt (300 mM NaCl) treatment. These proteins weredivided into the seven major categories (Fig. 6 and Table S2, ESI†).

Table 1 Mean and standard error of sodium (Na+), potassium (K+) and calcium (Ca2+) concentration (mmol g�1 dry weight) and the K+/Na+ ratio in barley leaves ofPiriformospora indica-colonized and non-inoculated plants under control (0 mM NaCl), moderate (100 mM NaCl) and severe salt stress (300 mM NaCl) conditions

Salt treatment (NaCl) Fungal treatment Na+ (mmol g�1 DW) K+ (mmol g�1 DW) Ca2+ (mmol g�1 DW) K+/Na+

0 mM Non-inoculated 1.30 � 0.08 34.94 � 0.98 5.73 � 0.34 26.87 � 0.53Inoculated 2.16 � 0.27 33.58 � 0.85 7.23 � 0.42 15.54 � 0.56




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In group A, eleven proteins were found which were reduced inboth the P. indica-colonized and the non-inoculated plants.These proteins included putative eukaryotic translation initia-tion factor 6 (protein spot 253), importin subunit alpha (proteinspot 827), Rubisco small chain (protein spots 41 and 884), Golgiassociated protein se-wap41 (protein spot 765), S-adenosyl-methionine synthase 1 (protein spot 691), nuclear transportfactor 2 (protein spot 8), fructokinase-2 (protein spot 527),

legumin-like protein (protein spot 549), translation initiationfactor (protein spot 166) and 6-4 photolyase (protein spot 817).In group (B) the abundance of 13 proteins which includedRubisco large subunit (protein spots 889, 946 and 947),Grx-S16-glutaredoxin subgroup II (protein spot 339), Mybfamily transcription factor (protein spot 338), profilin-1(protein spot 1), biostress-resistance-related protein (proteinspot 461), ankyrin repeat domain protein 2 (protein spot 814),

Fig. 4 Two-dimensional gel electrophoresis of control protein samples. The first dimension was obtained using 300-microgram (mg) proteins on linear gradient IPGstrips with pH 4–7. For the second dimension, 12% SDS-PAGE gels were used and the proteins were visualized using silver nitrate. The arrows represent protein spotsthat showed significant changes under salt stress as presented in Table 2.


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translational elongation factor EF-Tu (protein spot 606), chitinaseII (protein spot 681), chaperonin containing t-complex protein 1(protein spot 903), germin-like protein (protein spot 221) andputative mRNA binding protein (protein spot 665) weredecreased under 300 mM NaCl. Nonetheless, the abundanceof these proteins returned to control levels after P. indicainoculation and (C) protein cp31BHv was down-regulated onlyin P. indica-colonized barley plants. (D) Chloroplast oxygen-evolving enhancer protein 1 (protein spot 521), YLP (proteinspot 473) and fructan 6-fructosyltransferase (protein spot 791)

were down-regulated only in non-inoculated barley plants.(E) The abundance of three proteins including xyloglucanendotransglycosylase (protein spot 412), tubulin folding cofactorA (protein spot 95) and endotransglucosylase/hydrolase XTH2(protein spot 968) was increased only in P. indica-colonizedbarley plants. (F) The abundance of two proteins, Rubisco smallchain (protein spot 11) and papain-like cysteine protease(protein spot 413) were expressed and changed in oppositedirections in P. indica-colonized and non-inoculated plants.(G) The abundance of seventeen proteins was increased in both

Table 2 Proteins identified by MS (MALDI TOF/TOF)

Spot IDa

pI/MW (kDa)

Gene no.dScore/%Cov.e MS/MS-MSf Best matching gene productExp.b The.c

1 4.9/14 4.9/14.5 gi|1709779 324/32 4/3 Profilin-1 [Hordeum vulgare]8 5.9/13 6/13.6 gi|115477485 73/6 4/3 Nuclear transport factor 2 [Oryza sativa]11 6.1/13 8.8/18 gi|132107 213/46 11/3 Rubisco small chain [Hordeum vulgare]57 6.1/15 5.6/18.4 gi|56090131 546/57 7/5 Thaumatin-like protein TLP4 [Hordeum vulgare]66 5.4/15 9.7/19.7 gi|3914588 348/45 13/4 Rubisco small chain [Hordeum vulgare]95 5/18 4.7/12.9 gi|146760223 77/17 2/2 Tubulin folding cofactor A [Hordeum vulgare]117 4.5/18 4.3/11.2 gi|57471718 94/10 1/1 Ribosomal protein P1 [Triticum aestivum]119 4.6/18 6.2/23.3 gi|115444771 198/29 7/4 Peroxiredoxin-2E-2, chloroplastic [Oryza sativa]122 4.8/16 5.3/17.2 gi|115466344 139/18 8/4 Cytochrome c oxidase subunit Vb [Oryza sativa]125 5/19 4.8/17.7 gi|10799810 513/85 10/5 Cold-regulated protein [Hordeum vulgare]140 4.5/21 4.8/18 gi|125571152 161/23 4/3 Cytochrome c oxidase polypeptide Vb [Zea mays]166 5.7/20 5.1/12.6 gi|82754824 350/80 7/3 Translation initiation factor [Lophopyrum elongatum]205 6.7/25 7.6/26.8 gi|51039054 71/14 3/2 a-expansin EXPA2 [Triticum aestivum]219 6.6/27 6.7/28.3 gi|21954110 323/31 6/3 RNAse S-like protein [Hordeum vulgare]221 6.5/26 6/24.8 gi|1070358 145/30 4/2 Germin-like protein [Hordeum vulgare]247 4.6/29 4.9/30.7 gi|3550483 398/45 13/5 Cp31bhv [Hordeum vulgare]253 4.8/29 4.6/26.8 gi|115473637 155/11 2/2 Eukaryotic translation initiation factor 6 [Oryza sativa]301 6.1/25 5.9/22.9 gi|400094 151/32 10/6 23-KDa jasmonate-induced protein [Hordeum vulgare]325 5.5/26 9.5/27.4 gi|131394 555/51 10/6 Oxygen-evolving enhancer protein 2, chloroplast [Triticum aestivum]338 6/30 9.5/40.7 gi|4835766 77/33 12/0 Myb family transcription factor[Arabidopsis thaliana]339 5.9/29 9/31.4 gi|195655515 74/12 3/2 Grx_S16-glutaredoxin subgroup II [Zea mays]342 5.8/28 5.9/27.5 gi|15192923 78/21 4/2 Ascorbate peroxidase [Hordeum vulgare]352 5.5/27 5.3/26.9 gi|2507469 151/38 8/4 Triosephosphate isomerase, cytosolic [Hordeum vulgare]377 5.2/28 6.2/56.5 gi|14485487 636/33 14/6 Polyamine oxidase [Hordeum vulgare]412 4.7/33 4.6/31.5 gi|1890573 143/27 7/5 Xyloglucan endotransglycosylase (XET) [Hordeum vulgare]413 4.8/33 4.9/51.4 gi|194352754 119/7 3/2 Papain-like cysteine proteinase [Hordeum vulgare]441 5.4/31 9.7/19.7 gi|3914588 70/27 4/2 Rubisco small chain [Hordeum vulgare]447 5/17 4.9/17.3 gi|32765707 301/53 7/4 High light protein [Hordeum vulgare]461 6.4/30 9.7/34.3 gi|29409364 84/16 8/2 Biostress-resistance-related protein [Triticum aestivum]473 6.8/33 6.6/26.4 gi|4099148 226/59 14/4 Vacuolar proton-translocating ATPase subunit E [Hordeum vulgare]521 5.3/33 6/35 gi|147945622 529/46 12/5 Oxygen-evolving enhancer protein 1 [Leymus chinensis]527 5.2/38 4.9/35.8 gi|115474481 301/24 8/4 Fructokinase-2 [Oryza sativa]549 5.7/37 6.3/38.2 gi|162458978 117/14 4/3 Legumin-like protein [Zea mays]606 5.4/45 6.2/50.6 gi|17225494 114/21 7/1 Translational elongation factor Tu [Oryza sativa]621 4.9/42 5.3/46 gi|148763638 81/16 6/1 Magnesium chelatase 40-KDa subunit [Hordeum vulgare]636 5.8/32 6/31.7 gi|166064274 234/26 8/3 Xyloglucan xyloglucosyl transferase [Hordeum vulgare]665 5.9/39 8.8/41.3 gi|115471157 113/8 5/2 Putative mRNA binding protein [Oryza sativa]681 6/41 10/27.5 gi|9501334 344/22 7/6 Chitinase II [Hordeum vulgare]691 5.8/43 5.5/43.3 gi|68655435 161/43 13/2 S-adenosylmethionine synthase 1 [Hordeum vulgare]765 6/40 5.7/41.7 gi|162463414 156/29 11/3 Golgi associated protein se-wap41 [Zea mays]791 5.2/52 5.1/68.8 gi|60729576 340/20 10/6 Fructan 6-fructosyltransferase [Hordeum vulgare]814 4.7/51 4.5/37.3 gi|108712139 98/6 2/1 Ankyrin repeat domain protein 2 [Oryza sativa]817 4.7/51 10.1/67.6 gi|61816948 71/18 11/0 6–4 photolyase [Dunaliella salina]827 5.3/62 4.8/55.2 gi|218196127 81/16 6/2 Importin subunit alpha [Oryza sativa]884 5.6/83 5.8/13.3 gi|132107 102/35 3/2 Rubisco small chain [Hordeum vulgare]889 6.7/51 6.6/49 gi|133924511 147/15 9/4 Rubisco large subunit [Hordeum secalinum]903 6.2/62 6.1/61 gi|223531758 97/15 7/3 Chaperonin containing t-complex protein 1 [Ricinus communis]928 6/10 5.6/9.5 gi|108864033 137/57 4/2 Ubiquinol–cytochrome c reductase complex 7.8 kDa protein [Oryza sativa]946 6.6/49 6.6/49 gi|13310805 401/20 11/7 Rubisco large subunit [Hordeum secalinum]947 6.6/53 6.2/54 gi|31087921 512/42 22/7 Rubisco large subunit [Hordeum vulgare]968 4.8/32 5.8/29.3 gi|125570930 76/45 10/0 Endotransglucosylase/hydrolase XTH2 [Oryza sativa]

a The numbering corresponds to the 2-D gel in Fig. 4. b Experimental pI and MW. c Theoretical pI and MW. d gi number in Gene Bank. e MASCOTscore and percent coverage resulted from combined MS-MS/MS search. f Number of peptides identified by PMF and MS/MS.


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the non-inoculated and P. indica-treated barley plants under300 mM NaCl. These proteins included oxygen-evolving enhancerprotein 2 (chloroplast) (protein spot 325), polyamine oxidase(protein spot 377), triosephosphate isomerase (cytosolic)(protein spot 352), peroxiredoxin-2E-2, chloroplastic (proteinspot 119), Rubisco small chain (protein spot 66), xyloglucanxyloglucosyl transferase (protein spot 636), cold-regulatedprotein (protein spot 125), cytochrome c oxidase subunit Vb(122), 23 kDa jasmonate-induced protein (protein spot 301),a-expansin (protein spot 205), ascorbate peroxidase (proteinspot 342), thaumatin-like protein (protein spot 57), high lightprotein (protein spot 447), ubiquinol–cytocrome c reductasecomplex (protein spot 928), magnesium chelatase 40 kDa

subunit (protein spot 621) and RNase S-like protein (proteinspot 219). In this group, the abundance of the xyloglucanxyloglucosyl transferase and Rubisco small chain proteins weremore reduced in the P. indica-colonized plants (Fig. 6).

DiscussionP. indica enhanced barley adaptation to salt stress bymodulating the ion balance

In this study, the barley leaf sheath was used for proteomeanalysis, since the sheath is encased around the stem andexpresses signs of stress sooner than leaf blades. By analyzingthe physiological traits and molecular protein expression

Fig. 5 Abundance ratio of 18 individual barley protein spots showed statistically significant changes in response to salinity at both 100 mM and 300 mM NaCl inPiriformospora indica-inoculated (I) and non-inoculated (NI) plants compared to control (0 mM). The ratio is expressed as log2 (abundance in treated samples/abundance in control samples). Protein identification is extracted from Table 2. Proteins are grouped according to the expression pattern: (A) down-regulated in both Iand NI plants, (B) down-regulated only in NI plants at 100 mM salt stress, (C) up-regulated only in NI plants at 100 mM salt stress, (D) up-regulated only in I plants at100 mM NaCl, (E) up-regulated in both NI and I plants. The abundance ratio of the 18 mentioned proteins is also shown at 300 mM salt stress. Solid markers representa significant difference (p-value r 0.05) and open markers represent a non-significant difference (p-value > 0.05) in response to salt stress in P. indica-inoculated andnon-inoculated samples.


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changes indicated that the major aspects of the adaptivemechanism occurred in the P. indica-colonized barley plants.Salinity disturbs the plant nutrient relations by the availability,transport and partitioning of nutrients in plants. It also causesa deficiency, or imbalance of the ions Na+, K+ and Ca2+ due tothe competition for minerals. In addition, many plant pro-cesses such as growth, photosynthesis, mineral nutritionuptakes, water and ion transport are affected by the ratios ofthese ions.25

Moreover, the Na+ accumulation was remarkably lower inthe leaf sheaths of P. indica-colonized plants compared to thenon-inoculated barley plants (data not shown). In particular,the younger leaf sheaths accumulated less Na+ compared to theolder leaf sheaths of P. indica-colonized barley. This differencewas even more pronounced at a high salinity (data not shown).Although Na+ accumulation increased significantly in bothP. indica-colonized plants and non-inoculated plants under saltconditions, the foliar ratio of K+/Na+ was higher in P. indica-colonized plants compared with non-inoculated barley plants.

This ratio is a reliable indicator for plants with a high level ofsalt tolerance, which therefore indicates the adaptive mecha-nisms conferred by P. indica in colonized barley plants. K+ is acritical mineral nutrient that protects plants from salt-induceddamages26 and its impaired uptake by increased Na+ is well-documented in several plant species.27 This reduction could bedue to the negative effect of Na+ on K+ at root uptake sites,affecting transport into the xylem, or the inhibition of theuptake processes.28 It has been reported that salt-tolerantbarley plants maintain a lower amount of Na+ than non-tolerantones.29 Ca2+, which affects plant growth, photosynthesis, nutri-tion, water and ion transport, was greatly increased by thepresence of P. indica in colonized barley plants under controland especially under salt stress conditions. Many of theseresponses can be linked to the direct function of Ca2+ in theplasma membrane and the subsequent Ca2+ signaling events.P. indica affects intracellular Ca2+ signaling and is likely to impactthe stress signal transduction in Arabidopsis.30 It is probablethat this increase in Ca2+ concentration in P. indica-colonized

Fig. 6 Classification of 50 individual barley protein spots showed statistically significant changes in Piriformospora indica-inoculated (I) and non-inoculated (NI) barleyplants only at 300 mM NaCl condition compared with control. The ratio is expressed as log2 (abundance in salt-treated plants at 300 mM NaCl/abundance in controlsamples). Protein identification is obtained from Table 2. Proteins are grouped according to the expression pattern: (A) down-regulated in both NI and I plants,(B) down-regulated only in NI plants, (C) down-regulated only in I plants, (D) up-regulated only in NI plants, (E) up-regulated only in I plants, (F) modulated in both Iand NI, but in opposite directions, (G) up-regulated in both I and NI plants. Solid markers represent a significant difference (p-value r 0.05) and open markersrepresent a non-significant difference (p-value > 0.05) in response to salt stress in P. indica-inoculated and non-inoculated samples.


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barley plants not only affects mutualistic interaction but alsoinitiates the stress signal transduction leading to the increasedplant host adaptation to salt stress.

Salt-responsive proteins were altered in a similar direction inboth P. indica-colonized and non-inoculated barley plants

We found very few protein changes in abundance in response toP. indica-root colonization under control conditions (Table S3,ESI†). Some of them including quinone reductase 2, putative heatshock 70 KD protein (Hsp70), mitochondrial precursor, aspartate-semialdehyde dehydrogenase and Ribulose bisphosphatecarboxylase small chain were increased in P. indica- colonizedplants. In return, the abundance of actin and low temperature-responsive RNA-binding proteins were reduced in P. indica-colonized compared with non-inoculated barley plants undercontrol conditions. The quinone reductase 2 protein wasidentified based on the proteomics analysis in wheat andintroduced as the stress-responsive proteins. The aspartate-semialdehyde dehydrogenase is an important enzyme involvedin the biosynthesis of amino acids, which forms an early branchpoint in the metabolic pathway forming lysine, methionine,leucine and isoleucine from aspartate. The Hsp70 was anotheridentified protein increased in P. indica-colonized plant undercontrol conditions. Waller et al. (2009) showed that Hsp70 wasup-regulated in barley leaves of colonized plants, which can be auseful tool to analyze the systemic effects of fungal colonization.31

Hsp70 helps the proteins to maintain the tertiary structure andminimize the protein aggregation and degradation.

We observed that the abundance of several salt-responsiveproteins was changed in a similar direction in P. indica-colonizedand non-colonized plants (Fig. 5: groups A and E; Fig. 6; groups Aand G). The production of reactive oxygen species (ROS) isincreased under abiotic stresses, which leads to damage ofcells.32 In return, plant antioxidant systems prevent damageinduced by ROS and increased salt stress tolerance. A signifi-cant increase in the expression of ascorbate peroxidase (proteinspot 342) was observed in both P. indica-colonized and non-inoculated plants under salt stress conditions. Ascorbate per-oxidase is part of a plant’s enzymatic antioxidative defensesystems and a higher level of ascorbate peroxidase wasobserved in the salt-tolerant plant varieties.33 We also identi-fied polyamine oxidase (protein spot 377) as the salt-responsiveprotein, which was stimulated by salinity, and the expressionof polyamine oxidase was significantly affected by P. indica.Polyamines play a role in reducing the negative effects ofsalinity. Polyamine oxidase (PAO) is a source of apoplasticH2O2, and the salinity induces changes in plant polyaminelevels. ROS can modulate plant growth through a promotion ofcell wall cleavage. The involvement of PAO in the leaf growthand elongation in maize under salt conditions was shown.34

In accordance with the previous proteomics analysis, PAO washighly expressed in the salt-tolerant cultivar compared to theexpression in a salt-susceptible cultivar.35

Thaumatin-like protein (TLP) (protein spot 57) was anotheridentified salt-responsive protein in which the expression levelincreased in both the P. indica-colonized and non-inoculated

plants under severe salt stress conditions. TLP is associatedwith osmotic adaptation in plant cells36 and some TLPs areknown to protect plants against biotic and abiotic stresses suchas the pathogen infections, osmotic stress and freezing.37,38

One of the identified salt-responsive proteins with a higherabundance in both colonized and non-inoculated barley plantswas cytosolic triosephosphate isomerase (TPI) (protein spot 352).TPI is an important enzyme for carbon metabolism, especially inglycolysis and sugar metabolisms. The TPI activity increased thecarbon metabolism through the pentose phosphate pathway,CO2 uptake and the catabolism of sucrose to CO2.39 Proteomicanalysis revealed that TPI showed an increase in the expressionunder drought stress in maize.40 Over-expression of TPI showeda significant improvement of photosynthesis due to an increasein the rate of regeneration of Rubisco activity in transgeniccells.41

The most up-regulated identified protein in both P. indica-colonized and non-inoculated plants was an RNase S-likeprotein (protein spot 219). The amino acid sequence of anRNase S-like protein does not necessarily have S-RNase activityalthough it is closely related to RNase. It has been reported thatthe abundant S-like RNase protein was altered in response tothe various environmental stresses.42 Up-regulation of thisprotein in response to salt stress exhibits the impact onsenescence or nutrient deficiency processes that is induced bysalt stress.33,43 However, exactly why the physiological functionof noncatalytic S-like RNase was up-regulated under stressconditions is unclear. Further studies are needed to elucidatethe exact function of the RNase S-like protein specifically undersalt stress conditions.

An increase in the expression of ribosomal protein P1 wasobserved in both P. indica-colonized and non-inoculated plantsunder severe salt treatment. However, the expression increasedonly in P. indica-colonized plants under 100 mM NaCl treat-ment. Similar to the previous report, the ribosomal protein P1was reported as a salt-responsive protein in which the expres-sion level was increased in both the salt-tolerant and thesalt-susceptible barley cultivars.35 Salinity also induced theexpression of Magnesium chelatase (protein spot 621). Magne-sium chelatase is an enzyme involved in the process, whichregulates the chlorophyll biosynthesis. This protein is identi-fied as an abscisic acid (ABA) receptor in Arabidopsis.44 The ABAlevel was elevated in barley leaves under salinity;45 and there-fore, we expected that salinity mediates the expression of anumber of ABA-responsive proteins; however, expression ofABA related proteins was not altered in P. indica-colonizedbarley plants under salt stress conditions. The phytohormoneABA played a role in the P. indica-root colonization in barley.32,46

Nevertheless, a direct relationship between stress tolerance andincreased levels of ABA has not always existed.47

P. indica modulated barley proteome in response to differentlevels of salinity

The proteome response of P. indica-colonized barley plantswas less than the response in non-inoculated plants undersevere salt stress (45 protein spots versus 33 protein spots in


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non-inoculated and P. indica-colonized plants under 300 mMNaCl, respectively). Moreover, the trend of proteome change inthe P. indica-colonized and non-inoculated plants was different.P. indica-colonized barley plants showed mostly increase inabundance (21 and 12 proteins were up- and down-regulated,respectively). However, the major response in non-inoculatedplants was down-regulation under salt stress (19 and 26 proteinswere up- and down-regulated, respectively). Aside from the physicaland physiological impacts of P. indica, proteomic analysis revealedthat P. indica-root colonization resulted in the alteration of some ofthe mechanisms involved in salt-tolerance such as photosynthesis,cell antioxidant defense, programmed cell death, protein synthesisand cell wall arrangement in barley leaves.

Under a low level of salinity, the three down-regulatedproteins in non-inoculated plants showed no significantchanges in the P. indica-colonized plants (Fig. 5: group B).However, these proteins showed a similar response to a highlevel of salt in P. indica-colonized and non-inoculated plants.These proteins, nuclear transport factor 2 (protein spot 8),translation elongation factor EF-Tu (protein spot 606) andRubisco small chain (protein spot 884), are involved in majorcell metabolisms such as translation and photosynthesis.Furthermore, seven proteins (Fig. 5, group D), which showedno changes in the abundance in response to a low level ofsalinity, were changed significantly in P. indica-colonized plantsat a high level of salinity. A 23 kDa jasmonate-induced proteinwas identified in this group. This protein might be involved insystemic resistance against plant pathogens. The induction ofsystemic resistance in barley after P. indica-root colonization isdocumented and is similar to induced systemic resistanceactivated by a group of a root-associated non-pathogenicbacteria which act through the jasmonic acid (JA) signalingpathway.48,49 It is shown that salinity induced the JA responsein barley.50 In our study, the abundance of peroxiredoxins-2E-2(protein spot 119) increased only in P. indica-colonized plantsunder 100 mM NaCl. However, the abundance of this proteinincreased in both P. indica-colonized and non-inoculated plantsunder 300 mM NaCl, but to a higher extent in P. indica-colonized plants. Peroxiredoxin is a universal family of anti-oxidant enzymes and a new component in the antioxidantdefense network.51 It also controls the cytokine-induced per-oxide levels and thereby mediates the signal transduction. Thechanges in the abundance of peroxiredoxins in response tovarious stresses have been previously reported.43,52,53

By increasing the level of salinity to 300 mM NaCl, a numberof salt-responsive proteins were up-regulated in both P. indica-colonized and non-inoculated proteins. P. indica specificallyincreased the expression of several proteins involved in photo-synthesis under different salt stress conditions compared withnon-inoculated plants. The down-regulation of photosyntheticmachinery is a known phenomenon under salinity.42,54

Rubisco, which is involved in the first major step of carbonfixation, remained unchanged in P. indica-colonized barleyplants, but was significantly down-regulated in non-inoculatedplants under salinity stress conditions (protein spots 889, 946and 947). The Rubisco small chain (RBCS) (protein spot 11) was

highly expressed in P. indica-colonized barley plants, butsignificantly reduced in non-inoculated plants under salinestress conditions (Fig. 6). Two of the identified RBCS (proteinspots 441 and 884) were reduced in both the P. indica-colonizedand non-inoculated plants under salt stress conditions.

Salt stress affects the photosynthetic rate, due to this effectwe hypothesized that P. indica’s strategy to improve stresstolerance can be partly explained by maintaining photo-synthesis rates under salt conditions in order to prevent thesalt-induced damage. This is in agreement with the higherfresh and dry shoot weights of colonized barley plants com-pared with non-inoculated plants under both the control andsalt conditions.

Salinity hinders the plant growth by inhibition of shoot-growth, and therefore it is not surprising that salinity alters theexpression of proteins involved in cell wall biosynthesis and cellelongation processes. Several proteins involved in cell wallsynthesis or modifications were differentially accumulated inresponse to salt stress and P. indicia-root colonization. Theincluded proteins involved in cell wall biosynthesis and cellelongation processes such as xyloglucan endotransglycosylase(XET) (protein spot 636), tubulin-folding cofactor A (proteinspot 95) and a-expansin (protein spot 205). The abundance ofXET was increased in P. indica-colonized barley plants underdifferent salt stress conditions, but the expression remainedunchanged in non-inoculated plants under 300 mM NaCltreatment. XET has wall-loosening activity in plants and hasbeen involved in many aspects of the cell wall biosynthesis.55

In barley, the expression of XET was induced in response to theexposure of gibberellic acid (GA) and was stimulated during thecell elongation and leaf expansion.56 It was shown that GAbiosynthesis and signaling were activated in barley duringP. indica-root colonization.46 An additional cell wall-relatedidentified protein, tubulin-folding cofactor A was required forplant cell division, but not cell growth.57 It was up-regulatedupon exposure to different salt stress conditions (100 mM and300 mM NaCl) in colonized barley plants, but the expressionremained unchanged in non-inoculated plants.

The abundance of profilin (protein spot 1) decreased due toa high salinity in non-inoculated plants, but no significantprotein expression differences were observed in P. indica-colonized plants. Profilins are low molecular weight ubiquitousproteins, which bind to the actin monomer and cause cyto-skeleton remodeling, resulting in either the polymerizationor depolymerization of actin filaments. The cytoskeletonremodeling induces a spatial and temporal response of theplant cells to internal and external signals and is important forthe cell elongation and cell shape.58 Up-regulation of profilinmay be associated with cytoskeleton remodeling, which may berequired for the plant to adjust cellular behavior to substantialquantities of salt and, thereby, minimized the salt toxicity inplant cells.59

Another member of group B (Fig. 6) was identified as an Mybfamily transcription factor. This protein is involved in the severalaspects of the phloem development including the divisions ofthe phloem pole and sieve element differentiation.51 The loss-of


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function of this transcription factor hindered the developmentof the phloem sieve tube elements or companion cells.60

The abundance of papain-like cysteine proteases (proteinspot 413) decreased in non-inoculated plants under salt stress.However, P. indica-root colonization led to an increase ofabundance of this protein. Papain-like cysteine protease isinvolved in the cell signaling pathways, which plays an essentialrole in the plant senescence and programmed cell death and inresponse to the biotic and abiotic stresses.35 P. indica-rootcolonization is associated with the biotrophic phase and celldeath-dependent phase.8 P. indica-induced cell death is mole-cularly and biochemically different to necrotrophic cell death,which has already been implicated by the absence of root-necrotization in P. indica-colonized roots.58 Providing moreinformation about the function of papain-like cysteine proteasesmight provide a new insight into enhancing host tolerance tosaline conditions.

Concluding remarks

The potential application of symbionts in agriculture is notonly attributed to their ability to enhance crop yields but also isrelated to their ability to enhance tolerance to different abioticstresses. Plant symbionts offer a long-term abiotic stress toler-ance through host adaptation to environmental stress. In thisrespect, a deep understanding by which symbionts protect theirplant hosts against the detrimental effects of soil salinity mayprovide new insights into the stress adaptation mechanisms inplants and assist in helping design a better strategy to copewith abiotic stresses. According to our results, it is likely thatP. indica might induce systemic response to salt stress bychanging physiology and proteome of P. indica-colonized barleyplants, despite the fact that the fungus is not colonizing plantleaves. P. indica has a clear advantage with no obvious sideeffects for biotechnological applications. P. indica targets a fewsalinity host components over the higher manipulation of thehost in control plant under salinity. P. indica may also serve as amodel system to study molecular traits affecting abiotic resis-tance in cereals.61

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rsc mb c3mb70069k 3.a department of systems biology, agricultural biotechnology research institute...

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