Proteome, salicylic acid, and jasmonic acid changes in
cucumber plants inoculated with Trichoderma
asperellum strain T34
Guillem Segarra1, Eva Casanova1, David Bellido2, Maria Antonia Odena2,Eliandre Oliveira2 and Isabel Trillas1
1 Departament de Biologia Vegetal, Facultat de Biologia, Universitat de Barcelona, Barcelona, Catalonia, Spain2 Plataforma de Proteòmica, Parc Científic de Barcelona, Serveis Científicotècnics,
Universitat de Barcelona, Barcelona, Catalonia, Spain
Trichoderma spp. is one of the most commonly used biological control agents against plantpathogens. This fungus produces changes in plant metabolism, thus increasing growth andenhancing resistance to biotic and abiotic stresses. However, its modes of action remain to bedefined. In the first hours of interaction between cucumber plant roots and Trichoderma asper-ellum strain T34, salicylic and jasmonic acid levels and typical antipathogenic peroxidase activityincrease in the cotyledons to different degrees depending on the applied concentration of thefungi. The use of 2-DE protein profiling and MS analysis allowed us to identify 28 proteins whoseexpression was affected in cotyledons after cucumber root colonization by Trichoderma applied athigh concentrations: 17 were found to be up-regulated while 11 were down-regulated. Proteinsinvolved in ROS scavenging, stress response, isoprenoid and ethylene biosynthesis, and in pho-tosynthesis, photorespiration, and carbohydrate metabolism were differentially regulated by Tri-choderma. The proteome changes found in this study help to give an understanding of how Tri-choderma-treated plants become more resistant to pathogen attacks through the changes inexpression of a set of defence-oriented proteins which can directly protect the plant or switch themetabolism to a defensive, nonassimilatory state.
Received: February 15, 2007Revised: June 25, 2007
Accepted: July 25, 2007
Keywords:
Plant / Systemic resistance / Trichoderma
Proteomics 2007, 7, 3943–3952 3943
1 Introduction
The widespread soil fungus Trichoderma spp. is one of themost commonly used biological control agents against plantpathogens, although its modes of action remain to be estab-
lished [1]. The main mechanisms of disease control weretraditionally believed to be those that act primarily on patho-gens such as mycoparasitism, antibiosis, and competition forresources and space [2]. However, more recent data have ledto a new understanding of the way in which Trichoderma spp.interact with plants: this fungus produces changes in plantmetabolism, thus increasing growth and enhancing resist-ance to biotic and abiotic stresses [3–5].
Two major pathways are involved in the induction ofsystemic plant resistance: systemic acquired resistance(SAR) and induced systemic resistance (ISR). The first leadsto the expression of pathogenesis-related proteins, is salicylicacid (SA)-dependent and was initially described as beingpathogen inducible [6]. The second was initially described inplants colonized by nonpathogenic rhizobacteria, is jasmo-nic acid (JA)/ethylene (ET)-dependent and induces a primed
Correspondence: Guillem Segarra, Departament de BiologiaVegetal, Facultat de Biologia, Universitat de Barcelona, Diagonal645, 08028 Barcelona, Catalonia, SpainE-mail: [email protected]: 134-93-4112842
3944 G. Segarra et al. Proteomics 2007, 7, 3943–3952
state which enhances defence gene expression in the plantupon subsequent pathogen attack [7]. According to recentworks, the JA and ET dependency of ISR is based onenhanced sensitivity to these hormones, rather than on anincrease in their production [8].
In the particular case of Trichoderma asperellum strainT203 induction of plant defence, it has been shown that SAdoes not increase when applied at 105 germinated spores permilliliter, while JA and ET may be involved because the con-trol effect is diminished when their synthesis is inhibited.Furthermore, no changes were observed in ETconcentration.However, in contrast to typical ISR, peroxidase and chitinaseactivity and RNA expression of these genes increased tran-siently both systemically and locally (and only locally for b-1,3-glucanase) after the plant was inoculated with T203 inthe absence of pathogens [3, 4, 9].
Proteomics is becoming an increasingly important toolbecause proteins are directly related to function [10]. The highresolution of 2-DE makes it a powerful tool for separatingcomplex protein mixtures. This methodology has beenemployed to analyze plant proteins in response to both biotic[11] and abiotic stress [12]. The use of proteomics in biocontroland plant defense has been reviewed [13]. However, little isknown about the induction of protein or gene expressionchanges in the plant by Trichoderma spp. root colonization [14].
To further explore the effects of Trichoderma plant rootcolonization on systemic tissue and how it can affect theplant’s ability to defend itself against pathogens, we studiedhormone (SA and JA) and proteome changes produced inthe cotyledons of cucumber plants when grown in a liquidmedium inoculated with different concentrations of the bio-logical control agent T. asperellum strain T34, which has beenfound to be effective in reducing Fusarium wilt and Rhi-zoctonia damping-off in various plant species [15, 16].
2 Material and methods
2.1 Plant material
Cucumber seeds (Cucumis sativus L. cv. Negrito) were placedin 35 mL-multipots filled with perlite (mineral substrate) andgerminated in a chamber at 25 6 17C, with a photoperiod of16 h light at 200 mmol m22?s21. Eight-day-old cucumberplants were rinsed with tap water to remove perlite from theroots and placed on a perforated plastic sheet allowing rootsto grow freely in 3000 mL opaque plastic boxes containingaerated nutrient solution. The nutrient solution consisted of0.5 g/L Peter’s foliar feed 27-15-12 (Scotts, Heerlen, Nether-lands), 0.22 g/L CaCl2 and 0.25 g/L MgSO4?7H2O. Aerationwas provided by a small air compressor.
2.2 Plant inoculation with T34
T. asperellum strain T34 was grown in an agitated liquid me-dium containing 10 g/L malt for 5 days at 307C. The culture
was filtered to remove the mycelium and centrifuged at10 0006g (47C). The pellet was washed twice in distilledwater to obtain medium-free conidia. Conidia suspensionwas added to the nutrient solution to obtain different finalconcentrations of T34: noninoculated control, 105, 106, and107 conidia per milliliter. These four treatments will be refer-red as control, 105, 106, and 107. Concentrations were adjust-ed according to conidia counts in a hemocytometer.
2.3 Pseudomonas syringae pv. lachrymans (Psl)
inoculation
The foliar pathogen Psl was cultured overnight in liquidKing’s medium B [17] at room temperature, collected bycentrifugation, and resuspended in 10 mM MgSO4.Twenty-four hours after T34 treatment at 107 conidia permilliliter, cotyledons from 9-day-old cucumber plants wereinoculated with Psl following a protocol adapted from apreviously existing one [4]. Autoclaved sand (25 mg,0.3 mm particle size) and 100 mL of a bacterial suspension(OD660 = 1) containing 0.01% v/v surfactant (Tween 20)were applied to the surface of each cotyledon and gentlysmeared with the thumb while using disposable gloves.Controls were treated the same way with a 10 mM MgSO4
buffer without bacteria.To quantify the protective effect of T34 treatment against
Psl, multiplication of the pathogen was assessed 48 h afterchallenge. For each treatment, five randomly selected cotyle-dons were homogenized in a sterile solution of 10 mMMgSO4. Dilutions were plated onto King’s B agar supple-mented with 100 mg/L cycloheximide. After incubation at257C for 2 days, the number of Psl colony forming units pergram of tissue was determined. This experiment was per-formed three times.
2.4 Evaluation of peroxidase activity
After being transferred to the hydroponics system the coty-ledons of five plants per treatment were pooled per triplicateat different time points: 1, 3, 6, 12, 24, and 48 h. Plant sam-ples were ground under N2 and the resulting powder wasused to detect peroxidase activity as previously described [3].The ground matter was homogenized in phosphate buffer.The homogenate was centrifuged and the supernatant wasused to perform the assays. The peroxidase activity wasdetermined spectrophotometrically with phenol red as asubstrate.
2.5 SA and JA quantification
The ground samples were also used to determine SA and JAas described previously [18]. Samples were extracted withmethanol/water/acetic acid (90:9:1 by vol.). The extracts wereevaporated, reconstituted in the mobile phase and injectedinto the LC-ESI-MS/MS system in MRM mode.
Control and T34-inoculated plants at 107 conidia per millilitersampled 24 h after inoculation were chosen for proteomeanalysis. Three biological replicates of each treatment wereanalyzed (Figure 1 of Supporting Information). Groundplant samples (1 g) were extracted with 2 mL 0.2 M Tris-HClpH 7.9 buffer. The extract was centrifuged for 5 min at15 0006g. The supernatant was collected, filtered through anylon mesh (50 mm), mixed with 5 mL chilled TCA (20% inacetone) and left overnight at 2207C. The mix was cen-trifuged for 5 min at 15 0006g. The protein pellet obtainedwas washed with chilled acetone and dried under N2. Proteincontained in the pelled was quantified using a protein assaykit (BioRad Protein Assay Kit). An amount of 300 mg wasresuspended in 450 mL rehydration solution containing 7 Murea, 2 M thiourea, 2% w/v CHAPS, and 0.5% v/v IPG bufferpH 3–11 NL (Amersham Biosciences), 80 mM DTT, and0.002% of bromophenol blue and loaded onto 24 cm, pH 3–11 NL IPG strips [19]. IEF was performed using an IPGphorinstrument (Amersham Biosciences) according to manu-facturer’s instructions (active rehydration at 50 V for 12 hfollowed by a linear gradient from 500 to 8000 V until45 000 V/h). Focused strips were equilibrated for 15 min withequilibration buffer I (65 mM DTT, 50 mM Tris-HCl, 6 Murea, 30% glycerol, 2% SDS, bromophenol blue) and then for15 min with equilibration buffer II (135 mM iodoacetamide(IAA), 50 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS,bromophenol blue) [20]. The equilibrated strips were directlyapplied to 12.5% acrylamide gel [21] and separated at 3 V/gelfor 30 min and at 18 V/gel for 6 h in an Ettan DALT II system(Amersham Bioscience). After electrophoresis, proteins werestained overnight with a solution containing 0.1% w/v CBBR-250, 10% v/v acetic acid and 45% v/v methanol. After thewashing steps with 7% acetone, the gels were digitalyzedusing a scanner (ImageScanner from Amersham Bio-sciences). Images were analyzed with ImageMaster 2D soft-ware v. 4.01 (Amersham Biosciences) for detecting andobtaining volume values of spots present in gels. Normalizedvolumes were calculated by dividing the volume of each spotby the total volume of all spots in the gel and multiplying by ascaling factor. Normalized volumes of the spots detectedfrom three replicas of both control and T34-inoculated wereanalyzed in an ANOVA test using SPSS 14.0 statistical soft-ware.
2.7 In-gel digestion
In-gel digestion was performed automatically (Progest,Genomic Solutions) using the method described previously[22] with modifications. Briefly, selected spots were excisedfrom the gel with a razor blade. Excised gels spots werewashed sequentially with ammonium bicarbonate bufferand ACN. Proteins were reduced and alkylated, respectively,by treatment for 30 min with 10 mM DTT and 55 mM IAA.After sequential washings with buffer and ACN, proteins
were digested overnight at 377C with 0.27 nM trypsin. Trypticpeptides were extracted from the gel matrix with 10% formicacid and ACN; the extracts were pooled and dried in avacuum centrifuge.
2.8 Protein identification by MS
Tryptic digested peptide samples were analyzed by MALDI-TOF/TOF MS (4700 Proteomics Analyzer, Applied Biosys-tems) and/or ESI-MS/MS (Q-TOF Global, Micromass-Waters).
2.8.1 MALDI-TOF/TOF-MS
The digests were redissolved in 5 mL 0.1% TFA in 50% ACN.Typically, a 0.5 mL aliquot was mixed with the same volumeof a matrix solution, 2 mg/mL CHCA (Waters) in 0.1% TFAin 50% ACN and directly spoted on an MALDI-plate. MSspectra were acquired in positive reflector mode (voltage of20 kV in the source 1 and laser intensity ranged from 5800 to6200). Typically, 500 shots per spectrum were accumulated.Three major peaks were selected to be further characterizedby MS/MS analysis. MS/MS spectra were acquired usingCID with atmospheric air as the collision gas. A MS-MS 1 kVpositive mode was used. MS and MS/MS spectra from thesame spot were merged in a single mgf file prior to submis-sion for database searching.
2.8.2 ESI-MS/MS analysis
The tryptic digested peptide samples were analyzed by on-line LC-MS/MS (Cap- LC-nano-ESI-Q-TOF) (CapLC, Micro-mass-Waters). Samples were resuspended in 15 mL 1% for-mic acid solution and 4 mL were injected for chromato-graphic separation into a RP capillary C18 column (75 mm idand 15 cm length, PepMap column, LC Packings). Theeluted peptides were ionized via coated nano-ES needles(PicoTipTM, New Objective). A capillary voltage of 2000–3000 V was applied together with a cone voltage of 80 V. Thecollision in the CID was 20–35 eV and argon was used as thecollision gas. Data were generated in PKL file format andsubmitted for database searching.
2.8.3 Database searching
PKL and mgf files were submitted for database searching in aMASCOT search engine and PEAKS Studio v.3.1 againstSwiss-Prot, EMBL, and EST databases. The search parame-ters were: 1 missed cleavage, fixed, and variable modifica-tions were carbamidomethyl of cystein and oxidation ofmethionine, respectively. Peptide tolerence was 100 ppm and0.25 Da, respectively for MS and MS/MS spectra.
3946 G. Segarra et al. Proteomics 2007, 7, 3943–3952
3 Results and discussion
3.1 Induction of systemic resistance
T. asperellum strain T34 applied to the roots was able toreduce proliferation of the foliar pathogen Psl in cucumbercotyledons, indicating that induction of systemic resistancehad occurred (Fig. 1). Induction of systemic resistance bydifferent Trichoderma spp. has been previously described [1].
Figure 1. Multiplication of Psl in challenged cotyledons 48 h afterinoculation. Cotyledons of control and T34 plants were treatedwith inoculation buffer without bacteria. T. asperellum strain T34was applied at 107 conidia per milliliter to the roots of T34 andT34 1 Psl plants 24 h before challenge with the pathogen. Coty-ledons of Psl and T34 1 Psl plants were treated with inoculationbuffer containing bacteria. Values are means 6 SE from threedifferent experiments (n = 15). Values with different letters aresignificantly different (p,0.05) according to Tukey’s test.
3.2 Peroxidase activity and SA and JA measurements
The peroxidase activity in the cucumber cotyledons of controlsamples was between 0.20 and 0.34 mmol phenol red gramfresh weight21?min21 over the assay period (Fig. 2). Theperoxidase activity in the 105 treatment did not differ fromthat of the control. The peroxidase activity in the 106 treat-ment increased significantly at 12 h and gradually increasedto the maximum at 48 h after interaction with T34, in com-parison to the control and 105 treatments. The peroxidaseactivity in the 107 treatment increased significantly 6 h afterinteraction compared to rest of the treatments and reachedits maximum at 12 h. At 48 h there was no significant differ-ence between the peroxidase activity in the 106 and 107 treat-ments.
SA concentration in cotyledons of control samples wasbetween 13.8 and 77.8 ng/g fresh weight over the assay peri-od (Fig. 3A). The SA concentration in the 105 treatment didnot differ from that of the control over the studied period.The SA concentration in the 106 treatment was significantly
Figure 2. Time course of the peroxidase activity of cucumbercotyledons with or without T. asperellum strain T34 root inocu-lation at different concentrations. Values are means 6 SE (n = 3).Values with different letters are significantly different (p,0.05)according to Tukey’s test.
Figure 3. Time courses of the SA (A) and JA (B) content ofcucumber cotyledons with or without T. asperellum strain T34root inoculation at different concentrations. Values are means6 SE (n = 3). Values marked with asterisks are significantly dif-ferent (p,0.05).
higher at 24 h and higher still at 48 h after interaction withT34 in comparison to the rest of the treatments. The SAconcentration in the 107 treatment increased significantly3 h after the interaction reaching a concentration 20-foldhigher than that of the control and returned to control levelsat 24 h. If the 107 treatment is omitted from the statistics, theSA in the 106 treatment was significantly higher (2.5-foldincrease) than the others at 12 h.
JA concentration in cotyledons of control samples wasbetween 0.28 and 0.52 ng/g fresh weight over the assay peri-od (Fig. 3B). The JA concentration in the 105 and 106 treat-ments did not differ from that of the control. The JA con-centration in the 107 treatment increased significantly 1 hafter interaction reaching a concentration six-fold higherthan that of the control and returned to control levels at 6 h.
Taken together these results suggest that T34 effects onplant metabolism are dependent on concentration. At thelow concentration no effects on hormone or peroxidase ac-tivities were observed, which is consistent with typical ISR inthe case of root colonization by nonpathogenic rhizobacteria[23]. However, as stated in the introduction, this was not thecase of T203, which induces a transient increase of perox-idase activity and expression level of the gene in the leavesafter 72 and 48 h, respectively, after Trichoderma inoculationat 105 conidia per milliliter [3, 4]. No SA increase is observedin the leaves when T203 is inoculated at this concentration[4]. In our study we observed that when T34 is applied atmedium and high concentrations a SAR-like response isproduced with the typical concentration-dependent increasein SA and peroxidase activity. At the high concentration, thisSAR-like reaction may be sustained for at least 72 h (data not
shown) without affecting plant health or development. Al-though SAR has traditionally been associated with a directeffect of the inducer on the plant, it is now considered thatSAR inducers may play a dual role in the activation of plantdefence responses: low doses of SA prime for potentiatedinduction of certain defence genes after pathogen attack,whereas higher doses directly induce another set of defencegenes [24]. In addition, it is well known that JA levelsincrease rapidly and transiently in response to elicitor treat-ment [25, 26] and in a slower and sustained manner afterroot colonization by mycorrhizal fungi [27].
3.3 2-DE and protein identification
Using 2-DE, MS and database search, we analyzed thechanges in the proteome of cucumber plants inoculated withT34 at a high concentration (107 conidia per milliliter). Theresults obtained are shown in Fig. 4 and summarized inTable 1. The analysis of the images from all scanned gelsallowed us to discriminate 1011 different spots.
We analyzed the normalized volumes of the spots in anANOVA test (p�0.05) using the data from each of the threebiological replicates from control and Trichoderma inoculatedplants. Fifty-one spots were found to show significant differ-ences between treatments. Moreover, spots were consideredto be overexpressed if the ratio T34-normalized volume/Control-normalized volume was �1.5 and underexpressed ifthe ratio value was �0.66. Differentially expressed proteinswere selected to be analyzed by MALDI-TOF/TOF and/orESI-QTOF. The MALDI-TOF/TOF approach identified four
Figure 4. 2-DE protein profile (300 mg) of CBB-stained proteins from cucumber cotyledons grown in a hydroponic system containing con-trol nutrient solution (A) or T. asperellum strain T34-inoculated nutrient solution (B). Figures are one of the three biological replicates per-formed per treatment.
a) Assigned spot number as indicated in Fig. 4.b) Identified protein of C. sativus or homologous protein from other organism.c) The accession number code refers to (1) Swiss-Prot, (2) EMBL.d) All scores obtained from MASCOT, except *, which was obtained from PEAKS and is expressed as percentage (%).e) Theoretical pI.f) Theoretical MW.g) Means of three biological replicates 6 SD.h) p-Value obtained from ANOVA test including data from three biological replicates.
different gene products (spots 1569, 1156, 1054, and 1020).Other proteins were analyzed by the ESI-MS/MS method,which identified another 24 different gene products. Twoproteins (spots 1159 and 1145) could only be identified indbEST database. These information from dbEST identifica-tions allowed us to find homologies with other proteins byperforming Blast search against the nonredundant proteindatabases (Table 1).
Of these 28 identified proteins, 17 were found to be up-regulated in T34 treatment while 11 were down-regulatedcompared to control plants. The number of peptide matchesand the percentage of sequence coverage for each identifiedgene product are shown in Table 1. The identities of proteinswere deduced by similarity to available plant sequences.Most of the proteins identified showed spots focusing at dif-ferent pI or different apparent molecular weight, whichprobably correspond to differences in sequence and/orPTMs.
The proteins identified were classified into four groupsas described previously [28]. The first group includes pro-teins related to stress and defence, the second group includesproteins related to energy and metabolism, the third groupincludes proteins related to secondary metabolism and thefourth group includes proteins related to protein synthesisand folding (Table 1). Finally, several proteins remained uni-dentified. For some of these the MS and/or MS/MS signalswere good, but the results of the database search were nega-tive, probably because the sequences of these proteins are notyet included in public databases.
3.3.1 Disease and defence proteins
Of the proteins differentially expressed in this group, perox-idase is the most highly induced (30-fold) by inoculation withT34 compared to controls. This correlates with the highincrease in peroxidase activity observed in cotyledons of 107
T34-treated plants compared to controls. 2-Cys peroxir-edoxin-like protein was also up-regulated in T34-treatedplants, while peroxiredoxin, 2-cys peroxiredoxin, and cyto-solic ascorbate peroxidase were down-regulated. In normalphysiological conditions all of these antioxidant enzymes actas scavenging enzymes that remove ROS, thus protectingcells from oxidative damage. Biotic and abiotic stressesinduce an increase in ROS [29]. This accumulation can beviewed as threat to cells or as a secondary messengerinvolved in the stress-response signal transduction pathway[30]. If complete reduction does not occur, the result may be astate of oxidative stress leading to the oxidation of lipids,proteins, and DNA. Monodehydroascorbate reductase, whichwas found to be up-regulated in T34-treated plants, catalyzesthe reduction of an oxidized form of ascorbate [31]. SA isknown to inhibit ascorbate peroxidase activity [29] but we donot know whether this inhibition exists due to a reduction inprotein levels. In any case, we found cytosolic ascorbate per-oxidase to be down-regulated in T34 samples, which alsoshowed high levels of SA.
Aminocyclopropane-1-carboxylate (ACC) oxidase 1,which was found to be up-regulated in T34-treated plants, isinvolved in ethylene synthesis from ACC. In many cases,infection by microbial pathogens is associated withenhanced production of this hormone and a concomitantactivation of a large set of defence-related genes [32]. Duringinfection ACC accumulates transiently, which indicates thatACC oxidase activity restricts ethylene production [33]. Onthe other hand, it was demonstrated that the capacity toconvert ACC to ethylene was increased systemically in SAR-expressing Arabidopsis plants [8], thus providing a greatercapacity for ethylene production after challenge inoculation.
Heat shock protein 70 was found to be up-regulated.Heat shock proteins are cell chaperones that participate inthe folding and assembly of immature proteins, facilitateprotein transport and degradation of damaged proteins, andprevent the irreversible aggregation of denatured proteinsafter heat or other protein-denaturing stresses [34]. Recentfindings suggest that the HSP 70 family has a function indefence responses against pathogens [35].
In the plant proteome, specific pathogenesis anddefence-related proteins were also found to be associatedwith the interaction with T. atroviride [36] and T. harzianum[37].
3.3.2 Energy and metabolism proteins
We identified three spots that correspond to proteinsinvolved in the photorespiration metabolism, which hasbeen suggested to be important in maintaining electron flowto prevent photoinhibition under stress conditions [38]. T-protein is part of the glycine decarboxylase complex which isa key enzyme in photorespiration metabolism [39]. NADH-dependent hydroxypyruvate reductase is one of the mostactive photorespiratory enzymes that catalyzes the NADH-dependent reduction of hydroxypyruvate to glycerate [40].Formate-tetrahydrofolate ligase (formyltetrahydrofolate syn-thetase) (FTHFS) is one of the enzymes that participates inthe transfer of one-carbon units, an essential element of var-ious biosynthetic pathways. In many of these processes thetransfers of one-carbon units are mediated by the coenzymetetrahydrofolate (THF). In most eukaryotes the FTHFS ac-tivity is expressed by a multifunctional enzyme, which alsocatalyzes dehydrogenase and cyclohydrolase activity. Recentstudies in plants have shown that synthetase activity occursin a separate protein than the activity of cyclohydrolase andreductase, which occurs in a bifunctional protein [41]. For-mate is a potential alternative single-carbon source for theproduction of the 5,10-methylene-THF required for Ser syn-thesis, which occurs coupled to photorespiration and allowsthe continuous supply of THF [42].
Two spots corresponding to cytosolic glutamine synthe-tase were found to be up-regulated. This enzyme catalyzesthe ATP-dependent assimilation of ammonium produced inthe catabolism of amino acids into glutamine, using gluta-mate as a substrate. Interestingly, the RNA of this enzyme is
3950 G. Segarra et al. Proteomics 2007, 7, 3943–3952
also accumulated in tobacco plants inoculated with avirulentP. syringae but not with virulent bacteria; cytosolic glutaminesynthetase was also induced in leaf disks incubated with SA[43].
Two spots corresponded to proteins involved in main-taining the hexose-phospate pool. Glucose-6-phosphateisomerase, which was up-regulated in T34-treated plants,catalyzes the conversion of D-fructose 6-phosphate to D-glu-cose 6-phosphate. Cytosolic phosphoglucomutase (PGM),which was down-regulated in T34-treated plants, catalyzesthe conversion of D-glucose 6-phosphate to D-glucose 1-phosphate. Plants with strongly decreased expression ofPGM are characterized by a reduced photosynthetic rate [44].Fructose-1-6-bisphosphatase, which was also up-regulated, isa gluconeogenic enzyme that catalyzes the hydrolysis of offructose-1-6-bisphosphate to fructose-6-phosphate. Putativetransketolase, which is an enzyme involved in the pentosephosphate pathway, was up-regulated in T34-treated plants.This enzyme was shown to be important in protecting yeastagainst oxidative stress [45].
Enoyl-ACP reductase, which was up-regulated in T34-treated plants, is an important component of type II fatty acidsynthetase, which is responsible for the de novo fatty acidbiosynthesis in plants. Enoyl-ACP reductase has been relatedto both fatty acid synthesis for membrane and storage lipidsand to the systemic propagation of oxylipin signaling mole-cules such as jasmonate [46].
Two enzymes related to malate metabolism were foundto be up-regulated in T34-treated plants. NADP-malic en-zyme catalyzes the oxidative decarboxylation of malate toyield pyruvate, CO2, and NADPH. This cytosolic enzyme isinvolved in plant defence responses, possibly by providingNADPH for the biosynthesis of lignin and flavonoids [47].The promoter of the gene that codes for this enzyme in beanwas found to be activated by different effectors related toplant defence responses and agents that produce redox per-turbations [48]. Malate dehydrogenase, which was found tobe up-regulated in T34-treated plants, is involved in the oxi-dation of malate coupled to the reduction of NAD1, produc-ing oxaloacetate and NADH and therefore importing reduc-ing equivalents to the mitochondria in which the enzyme islocated. This enzyme was found to be up-regulated in differ-ent plants related to stress [49] or resistance against patho-gens [50].
Glyceraldehyde-3-phosphate dehydrogenase, which wasfound to be down-regulated in T34-treated plants, is an en-zyme involved in the glycoltic/gluconeogenic pathway. Thisenzyme is differently regulated under different stress condi-tions [51, 52].
ATP synthase beta-subunit, which was found to be up-regulated, is part of an enzyme that catalyzes the formationof ATP from ADP and Pi in the presence of gradients acrossthe membrane and has been found to be differently regu-lated in rice depending on the applied stress conditions [53].
NADPH-quinone reductase was up-regulated in T34-treated plants. This enzyme is related to the electron transfer
chain in plant mitochondrial membranes, although its pre-cise role is not clear. It was recently discovered that this en-zyme catalyzes the reduction of the toxic products generatedfrom lipid peroxides in Arabidopsis, thus acting as a sca-venger enzyme against oxidative stress [54]. NADPH-qui-none reductase gene was also found to be overexpressed inpotato during the defence response [55].
Tetrapyrrole biosynthesis is an essential pathway in ani-mals, plants, and microbes. The first common intermediateis 5-aminolevulinic acid (ALA). Porphobilinogen synthase,which was found to be up-regulated in T34-treated plants,catalyzes the formation of the tetrapyrrole precursor por-phobilinogen from two molecules of ALA. This pathwayleads to the biosynthesis of biologically important moleculessuch as heme, chlorophyll, vitamin B12, and cofactor F430[56].
We found rubisco activase to be down-regulated. Rubiscoactivase has been found to be up-regulated in plants underdrought stress [12], while in Arabidopsis under temperaturestress the opposite is observed [57]. We can expect to findrubisco activase to be down-regulated in a state in which theplant is switching to nonphotosynthetic-metabolism.Rubisco is maintained in an active state by the continuedaction of activase, a molecular chaperone that convertsrubisco from an inactive closed conformation to an activeopen conformation [58].
3.3.3 Secondary metabolism proteins
1-Deoxy-D-xylulose 5-phosphate reductoisomerase was up-regulated in T34-treated plants. This enzyme is involvedin nonmevalonate isopentenyl diphosphate biosynthesis,which is the fundamental unit in isoprenoid biosynthesis.Isoprenoid biosynthetic pathways have previously beenidentified as jasmonate inducible [59]. Although manyisoprenoids are related to plant defence, the transcriptlevels of this enzyme have also been found to beincreased in roots colonized by beneficial mycorrhizalfungi [60].
3.3.4 Protein synthesis and folding proteins
The translational elongation factor Tu, which was found to bedown-regulated in T34-treated plants, promotes the GTP-de-pendent binding of aminoacyl-tRNA to the A-site of ribo-somes during protein biosynthesis [61].
We found cyclophilin to be down-regulated in T34-trea-ted plants. Cyclophilins are ubiquitous proteins with anintrinsic enzymatic activity of petidyl-prolyl cis–trans isom-erase [62]. These proteins participate in the protein foldingprocess, not only as a prolyl isomerase but also as a chaper-one [63]. This protein has been found to be up-regulated indifferent cases of plant defence against pathogens andabiotic stress and also exhibits antifungal activity in vitro[64].
The results presented suggest that the induction of plantresponses by Trichoderma is a concentration-dependent phe-nomenon. In the first hours of interaction a reaction similarto SAR may occur in the cucumber plant, as a result of colo-nization by T34 when applied to the roots at a high con-centration. As a consequence, protection against a foliarpathogen is also observed. SA and JA levels peak and typicalantipathogenic proteins (i.e., peroxidase) are overexpresseddepending on the concentration of the fungi. Proteomicsprovides a convenient means of studying the plant-Tricho-derma interaction: 17 up-regulated and 11 down-regulatedproteins in T34 treatment compared to control plants wereidentified by the use of 2-DE protein profiling and MS anal-ysis of tryptically digested proteins. The proteins differen-tially regulated by T. asperellum strain T34 were involved inROS scavenging, stress response, isoprenoid, and ethylenebiosynthesis and in photosynthesis, photorespiration andcarbohydrate metabolism. Plants whose roots were colonizedby T34 overexpressed a group of defence-oriented proteinswhich are directly involved in protection or can switch themetabolism to a defensive, nonassimilatory state. Bydescribing proteome changes we are contributing to theunderstanding of how plants develop greater resistance topathogen attacks after root colonization by Trichoderma.
We thank the Departament d’Universitats, Recerca i Societatde la Informació of the Government of Catalonia and the Euro-pean Social Fund for the Ph.D. funding awarded to GuillemSegarra. This study was supported by the Spanish Ministry ofEducation and Science (AGL2005-08137-C03-01) and partiallysupported by ProteoRed. We thank Dr. Ilan Chet (The Weiz-mann Institute of Science, Rehovot, Israel) for kindly providingthe isolate of Pseudomonas syringae pv. lachrymans.
5 References
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