Trichoderma-Plant Root Colonization: Escaping Early Plant Defense Responses and Activation of the Antioxidant Machinery for Saline Stress Tolerance

Trichoderma spp. are versatile opportunistic plant symbionts which can colonize the apoplast of plant roots. Microarrays analysis of Arabidopsis thaliana roots inoculated with Trichoderma asperelloides T203, coupled with qPCR analysis of 137 stress responsive genes and transcription factors, revealed wide gene transcript reprogramming, proceeded by a transient repression of the plant immune responses supposedly to allow root colonization. Enhancement in the expression of WRKY18 and WRKY40, which stimulate JA-signaling via suppression of JAZ repressors and negatively regulate the expression of the defense genes FMO1, PAD3 and CYP71A13, was detected in Arabidopsis roots upon Trichoderma colonization. Reduced root colonization was observed in the wrky18/wrky40 double mutant line, while partial phenotypic complementation was achieved by over-expressing WRKY40 in the wrky18 wrky40 background. On the other hand increased colonization rate was found in roots of the FMO1 knockout mutant. Trichoderma spp. stimulate plant growth and resistance to a wide range of adverse environmental conditions. Arabidopsis and cucumber (Cucumis sativus L.) plants treated with Trichoderma prior to salt stress imposition show significantly improved seed germination. In addition, Trichoderma treatment affects the expression of several genes related to osmo-protection and general oxidative stress in roots of both plants. The MDAR gene coding for monodehydroascorbate reductase is significantly up-regulated and, accordingly, the pool of reduced ascorbic acid was found to be increased in Trichoderma treated plants. 1-Aminocyclopropane-1-carboxylate (ACC)-deaminase silenced Trichoderma mutants were less effective in providing tolerance to salt stress, suggesting that Trichoderma, similarly to ACC deaminase producing bacteria, can ameliorate plant growth under conditions of abiotic stress, by lowering ameliorating increases in ethylene levels as well as promoting an elevated antioxidative capacity.


Introduction
Trichoderma spp. are endophytic plant opportunistic symbionts widely used as biofertilizers and biocontrol agents for plant diseases [1,2]. Inhibition of plant disease severity is achieved through direct antagonism and mycoparasitism of the plant pathogens in the soil or on plant roots [3]. Although, some Trichoderma rhizosphere-competent strains can also colonize root intercellular spaces [4] and have direct effects on plants including increased growth and nutrient uptake, increased fertilizer efficiency utilization, percentage and rate of seed germination, and induced systemic resistance (ISR) to diseases [5]. Abiotic stresses frequently limit growth and productivity of major crop species, reducing yields to less than half of that possible under ideal growing conditions [6]. Trichoderma abilities to alleviate abiotic stresses are known, although specific knowledge of mechanisms controlling multiple plant stress factors is still lacking [7]. reported that Trichoderma harzianum T22 treated seeds germinated faster and more uniformly than untreated seeds whether the stress applied was osmotic, salt, or suboptimal temperatures. Application of the antioxidant glutathione, or application of T22, resulted in a similar positive effect. Recently [8], demonstrated that T22 improves tolerance to water deficit of tomato seedlings by enhancing the antioxidant defense mechanism and higher activity of ascorbate and glutathione-recycling enzymes. This finding thus supports the model that T. harzianum strain T22 increases seedling vigor and ameliorates stress by inducing physiological protection in plants against oxidative damage.
These data are comparable to the effects induced in plants by Piriformospora indica, a plant-root-colonizing basidiomycete fungus which can provide strong growth-promoting activity during its symbiosis with a broad spectrum of plants [9], and can induce resistance to fungal diseases and tolerance to salt stress that have been associated with an elevated antioxidative capacity [10][11][12]. Another mechanism for protecting plants against environmental stress has been demonstrated in plant growth promoting bacteria (PGPR), encoding the enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) (E.C. 4.1.99.4) that cleaves (ACC), the immediate precursor of ethylene [13]. A role for ACCD in the plant root growth-promotion effect by Trichoderma has been demonstrated by RNAi silencing of the ACCD gene in T. asperelloides showing decreased ability of the mutants to promote root elongation of canola seedling [14].
Within the last years, a few studies have reported on global transcriptome and proteome changes in plants colonized with Trichoderma. These studies led to the identification of genes and proteins that are likely involved in the beneficial interaction between Trichoderma and different plants species in root [15][16][17] and in leaf after the onset of induced systemic resistance (ISR) [18,19]. Recently, using microarray analysis [20] showed global gene expression changes in aerial part of Arabidopsis 24 hours after roots inoculation with Trichoderma harzianum T34. Most of the detected Arabidopsis defense-related genes, mediated by jasmonic acid (JA) and salicylic acid (SA), appear to be down-regulated by T. harzianum T34. Among these, genes involved in systemic acquired resistance (SAR) responses such as FMO1 (flavin monooxygenase 1) and PR-1 (pathogenesis-related 1), a marker for the SA dependent SAR response [21] are down-regulated. These findings suggest that Trichoderma, like the plant beneficial fungus Piriformospora indica, has to cope with plant defense responses during the initial stages of the interaction by broad-spectrum suppression of innate immunity, to allow colonization of Arabidopsis roots [22].
Induction of defense responses is typically associated with the coordinated transcriptional modulation of large numbers of genes [23]. WRKY DNA binding transcription factors have important roles in the regulation of genes associated with plant defense responses [24]. WRKY transcription factors function as positive and negative regulators of plant defense response has been demonstrated using Arabidopsis mutants [25,26]. A few studies also show the additive function of WRKY TF, demonstrating the weblike nature of the WRKY TF family [24]. For example, WRKY38 and WRKY62 have been shown to be negative regulators of plant basal defense in Arabidopsis [27]. Another example of additive mode of action of WRKY TF is the concomitantly action of WRKY18, WRKY40 and WRKY60 genes in Arabidopsis [28]. showed that WRKY18, WRKY40 and WRKY60 form homo-and hetero-complexes. When Arabidopsis were simultaneously mutated in WRKY18 and WRKY40 they gain better resistant to the biotrophic fungus Golovinomyces orontii [29], and to the hemibiotrophic bacterium Pseudomonas syringe [28]. On the other hand, WRKY18 and WRKY40 double mutants show higher susceptibility to the necrotrophic fungus Botrytis cinerea [28]. [30] showed that WRKY18 and WRKY40 negatively modulate the expression of positive regulators of defense such as CYP71A13, EDS1 and PAD4, but positively modulate the expression of some key JA-signaling genes by partly suppressing the expression of JAZ repressors. Hence, those TF are in the point of convergence between different defense signaling pathways.
In this study we follow the global gene expression in Arabidopsis roots colonized by Trichoderma and we show that 24 hours after the onset of colonization there are profound changes in plant transcripts associated to resistance to both biotic and abiotic stresses. Two main aspects of plant-Trichoderma interaction are highlighted by our analysis: plant gene modulation during root colonization by the endophyte and, tolerance to abiotic stresses conferred to the plant. Thus, by conducting further experiments using Arabidopsis mutant lines, cucumber and Arabidopsis plants exposed to salt stress with or without Trichoderma pre-inoculation, with Trichoderma wild-type or a ACC-deaminase gene mutant line, we provide evidence that: (i) during root colonization, Trichoderma manipulates WRKY18 and WRKY40 transcription factors activities to modulate the expression of the JAZ repressor genes and defense response genes, such as FMO1 and CYP71A13 to its advantage in the same way of biotrophic and hemibiotrophic plant pathogens (ii) Trichoderma fungi can ameliorate plant growth under abiotic stressful conditions by lowering deleterious elevated ethylene levels accompanied by an elevated antioxidative capacity.

Plant roots colonized by Trichoderma display pronounced changes in expression of transcripts involved in stress responses
To monitor global changes in genes expression following colonization of Arabidopsis roots by Trichoderma we performed microarray analysis 24 hours after the application of Trichoderma to the roots. Results indicate that Trichoderma root colonization affects the root transcriptome, with 249 probe sets showing an increased expression (.2 fold, p,0.0001) and 29 probe sets indicating a decreased expression (.2 fold, p,0.0001) after data filtering (File S1). Those 278 genes, showing significant change in expression, were subjected to Singular Enrichment Analysis (SEA) in order to identify enriched Gene Ontology (GO) terms [31]. Enriched functional categories were sorted according to statistically significant enrichment together with the gene identifier observed for each functional category (File S2). A substantial (False Discovery Rate (FDR),0.01), enrichment in genes correlated with response to biotic and abiotic stress (15% and 14% respectively, Table 1), response to different stimuli such as carbohydrate and chitin, as well as genes involved in hormone biosynthesis and response to hormone signaling, was detected. Interestingly, 7% of the total upregulated genes are related (FDR,0.01) to the oxylipin biosynthetic process (OPR3, AOS, OPCL1, LOX2, LOX3 and LOX4 genes), leading to JA biosynthesis (File S2, Table 1). A summary of the genes up-regulated in the microarray experiments with known role in plant hormone biosynthesis process and responses to hormone stimulation is presented in File S3.Among the down-regulated transcripts, SEA analysis did not reveal any significant enrichment

Author Summary
Trichoderma fungi have been developed as biocontrol agents and are applied to protect and improve crop yields. Colonization of plant roots by Trichoderma can protect plants against diseases and environmental stresses such as salinity and drought, and an improve plant growth and development. To better understand the mechanism underlining the plant-Trichoderma interaction we followed changes in global gene expression in colonized Arabidopsis roots. We associate the known gene biological function to the processes of root colonization and abiotic stress tolerance mediated by Trichoderma. Using Arabidopsis mutant lines we show the function of a subset of those genes in root colonization. We show that wrky18 and wrky40 transcription factors activate and suppress the expression of different genes in order to allow successful root colonization. We also combine the gene expression data together with the measurement of ascorbic acid level to demonstrate that salt stress tolerance offered by Trichoderma is dependent on activation of the plant antioxidant defense machinery. Using Trichoderma lines mutated in the ACC deaminase gene, we demonstrate that reduction of ethylene levels is also essential in achieving salt tolerance. This study represents an important step forward in understanding the nature of the non-pathogenic plant Trichoderma interaction, and may contribute to the efforts to improve Trichoderma biocontrol abilities.
in any class of GO-terms. Nevertheless noteworthy, were four members of the plant cytochrome P450 monooxygenases (CYP) family which were down-regulated. In Arabidopsis, CYP genes family includes 245 genes and constitutes up to 1% of the protein coding genes [32]. Co-expression analysis and data base search using STRING version 9.0 [33] and co-regulatory networks (ATTED-II; http://atted.jp/) did not, however, reveal any known correlation between those genes.Expression analysis was further performed using a profiling platform covering 137 biotic and abiotic stress responsive genes and transcription factors (List of genes and primers can be found in file S4). Transcript modulation by Trichoderma was followed by qPCR 9, 24 and 48 hours post inoculation (hpi). Heatmap representations ( Figure 1A and 1B) of significantly affected genes (p,0.05) reveal transient transcript activation during the first hours of interaction. Noteworthy, genes related to ethylene/JA regulation (WRKY41, WRKY53, WRKY55, ERF1, ERF6, ERF13 and RRTF1) ( Figure 1B), JA signaling (WRKY18, WRKY40, WRKY33, JIN1-7 and TDR1), root development and auxin modulation (MYB77), and secondary metabolites synthesis (MYB51). WRKY33 and MYB51 are also key elements of salt/osmotic stress regulation. Of note among other genes, mostly with function in general related defense responses and biotic and abiotic signaling process ( Figure 1A), are genes related to JA biosynthesis, LOX1, LOX3 and LOX4,and repressors of JA responses, (JAZ gene family) that show increased (JAZ5, JAZ6, JAZ7, JAZ8, JAZ9, and JAZ10) or decreased (JAZ11 and JAZ12) expression. Moreover, we observed an increase in expression of JA-responsive genes, such as VSP, VSP2 and PAD3. Increased expression of some of those genes such as WRKY18, JAZ8, JAZ10, LOX3 and LOX3 was also detected in the microarray analysis (File S1). Other Trichoderma modulated genes belong to the ethylenesignaling pathway, such as EIN2 and EIN4 which are positive regulators of ethylene responses. Auxin related genes include, ASA1 that function in jasmonate mediated regulation of auxin biosynthesis and lateral root formation and eir1 involved in auxin transport ( Figures 1A). As a large protion of the Trichoderma responsive genes correspond to processes directly associated with JA, Ethylene and Auxins metabolism and response, we zoom in on those differentially expressed genes to obtain an overview of the changes and their possible biological role in more detail ( Figure 2). Of note, increased expression was also observed in genes of the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL1 and PAL2) a key enzyme of the first step of the pathway and 4CL, which is involved in the last step of the pathway. Moreover, substantially large numbers of genes that take part in different defense response processes like PDF1.2, PR-2 and PR-4 are upregulated ( Figures 1A and 1B, File S1). The expression of 22 genes that show increase expression in the microarray analysis are in agreement with the qPCR results, thus validating the microarray analysis (File S5). Mapman software [34] analysis further highlight the biological processes affected during Trichoderma colonization ( Figure S1).
Root colonization by T. asperelloides is reduced in WRKY18 and WRKY40 double mutant knockout line Our expression analyses using both microarray and qPCR revealed significant up-regulation of WRKY18, WRKY40 and WRKY60 transcription factors (TF) as early as 9 hours from the onset of the interaction. Those TF genes encoding regulators of the JA signaling networks enable plants to coordinate and finetune responses to different pathogens [28][29][30]. To investigate whether WRKY18 and WRKY40 genes have a role in root colonization, we evaluated Trichoderma root colonization in wrky18/ wrky40 double knockout line and compared with WT Col-0 plants. Total DNA was extracted from roots at 12, 24, 48 and 96 hpi and the amount of Trichoderma fungal DNA was quantified using Trichoderma specific primers and normalized to plant reference genes [35]. wrky18/wrky40 line exhibited reduced root colonization ability (P,0.05, Figure 3A). To determine if one gene was functionally sufficient to restore WT level of colonization, and to define the contribution of WRKY40 in this process, we evaluated root colonization in a line were the WRKY40 was over-expressed in the wrky18/wrky40 background mutant [30]. Trichoderma colonization ability was clearly enhanced compared with the double mutant ( Figure 3A), but colonization did not reach WT levels (P,0.05). This indicates that both WRKY18 and WRKY40 functions are required, but that WRKY40 over-expression alone is sufficient to partially restore the colonization level of the WT plants.STRING analysis (version 9.0, [33]) was applied to generate a correlation network between WRKY18, WRKY40 and WRKY60 and other genes, based on co-expression, physical and functional interactions and public based knowledge ( Figure 4). Seven out of  twenty five genes of the network showed increased expression in the microarray analysis. This is a significant enrichment of upregulated genes in the network compared to the total number of up-regulated genes represented on the microarray chip (Fisher exact test, p = 2.207E-8). Moreover, by carefully monitoring the expression of all genes in the network using qPCR, we conclude that 12 genes show significant increased expression (p,0.05) 24 hpi (Figure 4). A subset of these 12 genes show also increased expression in time points 9 and 48 hpi ( Figure 1A and 1B).
WRKY18 and WRKY40 positively regulate JA signaling and negatively regulate the expression of FMO1, PAD3 and CYP71A13 defense genes in response to Trichoderma To gain better understanding of the role of WRKY18 and WRKY40 in T. asperelloides T203 plant root interaction, the expression of FMO1, PAD3 CYP71A13, AOS, LOX2, JAZ8 and JAZ10 which we found to be up-regulated in Arabidopsis WT during root colonization ( Figure 1A; File S1), was monitored in the wrky18/wrky40 double knockout lines inoculated with Trichoderma. each time point, and is relative to control collected in each one of the time points. Black: no significant difference (P.0.05), red-up regulation, greendown regulation significantly different from control (P,0.05). doi:10.1371/journal.ppat.1003221.g001 Figure 2. Overview of modulation of expression of phyto-hormone biosynthesis and responsive genes in Arabidopsis roots during Trichoderma colonization. In the early steps of the interaction, Trichoderma secreted MAMPs that triggered activation of signal transduction which modulated the expression of several genes. Among them, genes that have been previously shown to have a role in biosynthesis and response to JA, ethylene (Et; significant enrichment, FDR: P,0.01) and auxin (non-significant enrichment). Based on previously published data, we propose two main biological roles for the activation of those phyto-hormone related pathways in the plant-Trichoderma interaction: (i) colonization regulated by JA and ethylene, and (ii) lateral root formation regulated by JA and ethylene. Intermediate metabolites in the biosynthesis pathways are indicated by bold frame. Genes mediating the biosynthesis process are indicated without frame or marked in blue in case they show a significant (P,0.05) change in expression level upon Trichoderma colonization. Down-regulated genes are underlined. The model is based on the following published studies [28,30,[39][40][41]50,[76][77][78]  For FMO1, PAD3, and CYP71A13, we detected higher transcript levels in wrky18/wrky40 than in WT plants ( Figure 5A) suggesting that these genes, encoding a SAR regulatory gene and two key genes in camalexin synthesis are under negative control by WRKY18 and WRKY40 in WT plants. WT plants showed 3.8 and 2.8-fold elevated levels of LOX2 and AOS transcripts, respectively at 24 hpi. In contrast, Trichoderma-induced expression of both genes was completely absent in wrky18/wrky40 ( Figure 5B). These findings suggest that wrky18/wrky40 plants fail to induce the JA pathway. Transcript levels of the JAZ8 and JAZ10 genes were elevated in unchallenged wrky18/wrky40 plants ( Figure 5C) 9 hours post Trichoderma colonization, and their expression declined significantly over the colonization period. In the case of JAZ8, transcript reached similar levels to those in WT plants at 48 hpi ( Figure 5C). These results may indicate that the failure to induce the JA pathway in wrky18/wrky40 plants may be due to elevated levels of JAZ repressors, and therefore we can assume that WRKY18 and WRKY40 regulate expression of JAZ genes during root colonization.

Fmo-1 mutant knockout line shows increased root colonization by T. asperelloides
Our findings indicate that upon colonization of the root by Trichoderma, WRKY18 and WRKY40 transcription factors regulate the expression of the flavin-dependent monooxygenase1 FMO1 gene ( Figure 5A). To investigate whether FMO1 has a protective role during root colonization, we evaluated Trichoderma root penetration in FMO1 knockout line (fmo1) in comparison to WT Col-0 plants. Trichoderma mycelia were recovered from root tissues 24 and 48 hpi and at both time points an increased root colonization was detected in the fmo1 mutant ( Figure 3B). These results, together with data shown previously ( Figure 1A, 4 and 5A; File S1), support a model where activation of FMO1 upon Trichoderma root colonization is in part negatively regulated by the WRKY18 and WRKY40 to allow a moderate level of colonization.
Trichoderma root colonization induces changes in the level of indole glucosinolate metabolites We found that the expression of the MYB51 gene is induced 9, 24 and 48 hours after the application of Trichoderma ( Figure 1B) [36]. showed that indole glucosinolates (IGS) biosynthesis is regulated by MYB51, downstream ethylene signaling pathway, and demonstrated the role of 4-methoxy-indol-3-ylmethylglucosinolate (4-methoxy-I3G) in the Arabidopsis immune response.
The influence of Trichoderma on the level of IGS in Arabidopsis roots, 24 hours after colonization, was therefore tested by targeted LC-IT/ESIMS analysis. A significant increase in the level of 4methoxy-I3G and methoxy-3-indolyl-methylglucosinolate 1MI3MG-1 and decrease in the level of Indolyl-methyl glucosinolate (I3M) was observed (P,0.001; Figure S2).
Trichoderma root colonization increases the expression of antioxidant enzymes and the ascorbic acid pool during salt stress An increased expression of genes that function in general tolerance to abiotic stresses (File S1) was pointed out by the results of the microarray analysis. Among them, genes that take part in salt tolerance and osmoprotection processes (File S3). To further test Trichoderma-induced plant response to salt stress, the expression of 28 Arabidospis genes previously identified as NaCl responsive genes [37] and related to osmoprotection, or to general response to oxidative stress, was monitored by qPCR in control plants and plants subjected to salt stress (100 mM NaCl) with or without Trichoderma pre-treatment 48 hours before salt stress imposition ( Figure 6A). Worthy of note was the induced expression of MDAR, APX1 and GST indicating a general activation of the plant antioxidant machinery by Trichoderma. Activation of the antioxidant defense by Trichoderma was found also in cucumber (Cucumis sativus) seedlings subjected to salt stress (100 mM NaCl) with or without Trichoderma pre-treatment. The expression of the cat, sod(Mn) and sod(Cu) genes was analyzed by qRT-PCR, one and four days after salt addition ( Figure 6B). After 24 hours the expression of all tested genes was increased (5-8 fold) by the salt addition and also by Trichoderma root colonization. Enhanced expression of the genes could be detected in seedlings that were infected with Trichoderma 48 hours prior the salt addition (+T+NaCl). After four days ( Figure 6B) the cat gene expression is decreased in all treatments, while the expression of the sod (Mn) and sod(Cu) genes remains elevated in the plants under salt stress and even higher in the Trichoderma pretreated plants. In the absence of stress imposition, higher ascorbate levels in its reduced form were measured in Trichoderma treated cucumber plants, whereas levels of dehydroascorbate were reduced giving a higher AA/DHA ratio (Table 2). Accordingly, the relative MDAR expression level is increased by 15 fold in cucumber plants treated with Trichoderma for 48 hours, as measured by qPCR. Upon saline stress imposition in the presence of Trichoderma the levels of reduced and oxidized forms of ascorbic acid were comparable to those of control plants, confirming the protective effect of T. asperelloides T203.
Trichoderma wild-type but not ACC-deaminase silenced mutants can improve cucumber and Arabidopsis seed germination under salt stress conditions The ability of Trichoderma to ameliorate seed germination under saline stress conditions was assessed in soil. In absence of saline stress cucumber ( Figure 7A) or Arabidopsis ( Figure 7B), seeds planted in soil treated with Trichoderma T203 (WT) or with ACCdeaminase mutant spores showed similar germination rates to  those of the control seeds treatment. However, when cucumber ( Figure 7A) or Arabidopsis ( Figure 7B) seeds were treated with saline solutions, the germination rate was significantly higher in Trichoderma WT treated soil than in untreated (control) or ACCdeaminase mutant treated soil. These results indicate that the afforded tolerance to the salt application is probably linked to a lower level of deleterious ethylene evolving under stress conditions.

Discussion
Relatively little is known about the host mechanisms that connect the perception of Trichoderma root colonization to the downstream signaling pathways leading to activation of defense and developmental responses. It is assumed that microbeassociated molecular pattern (MAMP) recognition triggers the Figure 6. qRT-PCR expression analysis of antioxidant enzymes in cucumber seedlings and roots of four weeks old Arabidopsis under salt stress. (A) RNA was extracted from untreated Arabidopsis roots and from roots collected from plants exposed to 100 mM NaCl (NaCl), and from roots colonized by T203 for 48 hours prior exposure to 100 mM NaCl (T+NaCl). Hierarchical clustering by Euclidean distance method average linkage is shown. The color of the each cell indicates fold-change relative to control as follows: Red: significant up regulation (P,0.05; t test), green: significant down regulation (P,0.05; t test). Black: not statistically significant difference from the control,. (B) RNA was extracted from untreated cucumber seedlings (Control), from seedlings exposed to 100 mM NaCl (+NaCl), from seedlings colonized by T203 (+T) and from seedlings colonized by T203 for 48 hours prior exposure to 100 mM NaCl (+T+NaCl). RNA was extracted 1 day and 4 days after salt addition. Fold expression (for A and B) was calculated from average of three independent biological repetitions. doi:10.1371/journal.ppat.1003221.g006 activation of a signaling cascade that activates a variety of defense responses including callose deposition, programmed cell death, production and accumulation of antimicrobial reactive oxygen species, and induction of phytoalexins and other secondary metabolites [5].
In the current study, microarray analysis of Arabidopsis roots colonized by Trichoderma, coupled with qPCR analysis, allowed identification of genes involved in activation of the first stage of root colonization by the fungus, plant growth responses, biotic resistance and abiotic tolerance induced by this beneficial fungus.
Trichoderma effects on plant growth promotion and root architecture are well known [8,38]. Trichoderma enhanced biomass production and lateral root growth promotion were shown to be an auxin-dependent mechanism in Arabidopsis [39]. In the present study we found that MYB77, which is involved in auxin response and later root formation [40], and ASA1, that functions in jasmonate mediated regulation of auxin biosynthesis and transport during lateral root formation [41], are up-regulated during Trichoderma root colonization ( Figure 1A and 1B, Figure 2). Interestingly, microarray results indicate activation of other genes related to root and plant development. Among them are ATPSK2, involved in cell proliferation and organ morphogenesis [42,43], and the transcription factor ANAC081, known to be linked to increased leaf size and biomass traits [44].
Genes with known role in plant defense responses are upregulated during Trichoderma colonization (File S1), for example, PR-2 and PR-5. Interestingly, prominently four genes (PDF1, PDF1.2, PDF.1.2c, PDF1.3) out of thirteen members of the Arabidopsis defensin gene family, that inhibit the growth of a broad range of fungi, [45], are up-regulated. These findings are comparable to similar results obtained by [46].
Increase in transcript abundance of MYB72 ( Figure 1B) is in agreement with the results obtained by [47] which demonstrated the role of MYB72 in the early signaling steps of Trichoderma mediated ISR.
The ethylene dependent MYB51 which is involved in the transcriptional activation of indole glucosinolate (IGS) biosynthetic genes during plant defense responses [36] is transiently upregulated by Trichoderma with highest expression at 24 hours after the application of Trichoderma to the roots. An increase in the expression of CYP79B3, that take part in the conversion of tryptophan to indole-3-acetaldoxime a precursor of indole glucosinolates and the anti-microbial molecule camalexin, was observed too (Fig. 1A).
Because CYP79s catalyze a key step in the biosynthesis of glucosinolates, plant products that function in the defense toward herbivores and pathogens, alteration of the expression of these genes often has dramatic effects on the profile of glucosinolates. To further exploit the induction in the expression of MYB51 and CYP79s genes, the content of three key IGS metabolites was measured in Trichoerma treated and control Arabidopsis roots. Our results show a significant increase in the level of 4-methoxy-I3G and 1MI3MG-1 and decrease in the level of their precursor I3M ( Figure S2). Similarly [36], showed that Flg22 elicited biosynthesis of 4-methoxy-I3G, associated to MYB51 activity. Interestingly, two other CYP genes beside CYP79B3, CYP71B15 and CYP71A13, that function in camalexin biosynthesis [48], are significantly affected by Trichoderma root colonization ( Figure 1B).
Among the 28 transcripts down-regulated 24 hours after Trichoderma inoculation, four of them are plant cytochrome P450 monooxygenases (CYP712A2, CYP712A1, CYP93D1 and CYP76G1). These genes mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects [49]. Their down-regulation could indicate a strategy for repressing local defense responses to allow successful colonization as recently reported for P. indica [22,50]. This strategy is also supported by the induced expressions of the transcription factor ANAC081, which has been shown to be Cotyledons were sampled from seven days old seedlings grown in soil with or without Trichoderma (Control and +T203) with or without 75 mM NaCl (+NaCl and +T203 +NaCl).  a repressor of the expression of genes that encode pathogenesisrelated proteins, and the over-expression of this TF causes susceptibility to the soil-borne fungal pathogen Fusarium oxysporum [44]. Transcriptional activation upon pathogen attack is a common feature of most of the group III WRKY genes. Furthermore, most of these genes are expressed during both non-host and R genedependent resistance [51]. Three WRKY group III transcription factors (WRKY41, WRKY53, and WRKY55) are up-regulated during the first 24 hours of Trichoderma root colonization, but their expression goes down together with expression of other defense related transcripts, again supporting the idea that Trichoderma somehow can temporarily repress local defense plant immune response [20].
Trichoderma root colonization triggers also a rapid increase in transcription factors expression like WRKY18, WRKY40, WRKY60 and WRKY33, which activate JA-pathway responses and represses SA signaling. WRKY18, WRKY40 and WRKY60 are pathogeninduced and encode three structurally related WRKY proteins which exert a positive role in JA-mediated defense [28,30]. Trichoderma regulate the expressions of the WRKY18 and WRKY40 TFs to allow root colonization, in a similar way to the biotrophic fungus Golovinomyces orontii and the hemibiotrophic bacterium Pseudomonas syringe and in contrast to necrotrophic fungus Botrytis cinerea [28,29]. Hence, beneficial microorganism and plant pathogens share common molecular mechanism to cope with the plant immune system.
Interestingly, our findings resemble the interaction observed during the early stages of infection of Arabidopsis by G. orontii [30]. In the course of infection, G. orontii manipulate WRKY18 and 40 transcription factor activities to modulate the expression of JAZ repressor genes and defense response genes, such as FMO1 and CYP71A13 to its advantage. Recently [20], in microarray analysis of aerial parts of Arabidopsis plants 24 hours after application of T. harzianum T34 showed reduced expression of the genes FMO1 and CYP71A13. Although T203 colonization induces the expression of FMO1 and CYP71A13 in Arabidopsis WT (Col-0) plants ( Figure 1A, Figure 5A, File S1), monitoring those genes expression in wrky18/ wrky40 double knockout line showed a significant higher expression upon colonization by T203 ( Figure 5A). Hence, increased T203 colonization of the fmo1 knockout line ( Figure 3B) provides further evidence for a model were Trichoderma spp. fine-tune the expression of defense genes such as FMO1 and CYP71A13 to allow colonization.
FMO1, the flavin-dependent monooxygenase, was reported to be essential for the initiation of systemic resistance in Arabidopsis [52,53], although its deletion did not affect local defense responses at the site of pathogen attack. It was suggested that a metabolite generated by FMO1 might be necessary during the early phase of SAR establishment and that FMO1 contributes to a signal amplification loop required to potentiate SAR responses in systemic tissues. The involvement of FMO1 in Trichoderma mediated ISR cannot be excluded too. T-DNA insertion into the FMO1 gene resulted in enhanced susceptibility to virulent Pseudomonas syringae and Hyaloperonospora parasitica [54]. In our study, the same T-DNA insertion line revealed increased colonization by T. asperelloides ( Figure 3B), thus showing FMO1 function in defense response also in roots, and how its function is essential to balance the level of root colonization by T. asperelloides.
MYB51, MYB15 and WRKY33 are also key elements in the plant response to abiotic stresses. WRKY33 over-expression was found sufficient to increase Arabidopsis tolerance to NaCl and to regulate transcription of several downstream genes involved in the response to stress [55]. This and other molecular changes induced in the plant by Trichoderma root colonization are in good correlation with the protective effect induced in plants by these beneficial fungi towards a plethora of environmental stresses. Plants contain a series of enzymatic antioxidants such as superoxide dismutase (SOD), ascorbate peroxidase (APX; EC 1.11.1.11), and catalase. These antioxidants function properly to interrupt the cascades of uncontrolled oxidation in some organelles and to scavenge the toxic ROS produced under environmental stresses.
We could show that in cucumber the expression of cat and sod genes is affected by Trichoderma colonization of the roots resulting in long lasting up-regulation upon salt stress imposition ( Figure 6B). Besides these enzymatic mechanisms, low molecular mass antioxidants are efficient radical scavengers and may play a role in oxidative stress response of higher plants. Ascorbate is a major antioxidant that is involved in the ascorbate-glutathione cycle. Higher levels of ascorbate in its reduced form were found in Trichoderma treated plants (Table 2), similarly to data reported for plant root inoculation by P. indica [12]. Monodehydroascorbate reductase (MDAR) is the enzymatic component involved in the regeneration of reduced ascorbate. MDAR was shown to be crucial for the mutualistic interaction between Arabidopsis and P. indica [11]. Interestingly, this gene is also highly induced in cucumber and Arabidopsis during root colonization by Trichoderma ( Figure 6A, File S1). In Arabidopsis, Trichoderma pre-treatment prior salt stress imposition also stimulates expression of various transcripts involved in osmoregulation and general oxidative stresses ( Figure 6A). The mechanism proposed in this study according which T. asperelloides T203 can induce plant tolerance to salt stress has been proposed recently, also by [8] in relation to the tolerance afforded to tomato seedlings to water deficit by pretreatment with T. harzianum T22. In consequence, the activation of the antioxidant machinery in order to recycle oxidized ascorbate appears to be a general mechanism activated by different Trichoderma strains (T. asperelloides and T. harzianum) in different plant species (Arabidopsis, cucumber and tomato) to enhance tolerance to a range of abiotic stresses.
Beside the activation of the antioxidant machinery, the microarray results show also activation of other genes, which have protective role in Arabidopsis against salinity and osmotic stress (File S3). For example, we detected up regulation of two members of the aquaporin gene family (AT2G34390 and AT2G29870). Heterologous over-expression of rice and wheat aquaporin genes in Arabidopsis resulted in increase tolerance to salinity and dehydration [56,57].
Biosynthesis of ethylene in plants under salinity stress is well established. Higher ethylene concentration inhibits root growth and ultimately affects the overall plant growth. Many studies have shown that ethylene level in plants is regulated by a key enzyme 1aminocyclopropane-1-carboxylicacid (ACC)-deaminase. This enzyme present in plant growth-promoting bacteria (PGPR) and other microorganisms [58], lowers the ethylene level by metabolizing its precursor ACC into a-ketobutyrate and ammonia (NH 3 ).
Inoculation of plants under salinity stress with PGPR having ACC-deaminase activity mitigates the inhibitory effects of salinity on root growth by lowering the ethylene concentration in the plant. This in turn results in prolific root growth, which is beneficial for the uptake of nutrients and maintenance of growth under stressful environment [59].
In a previous work we presented data based on Trichoderma silenced mutants suggesting a central role for ACC deaminase (ACCD) activity in the plant growth promotion effect by T. asperelloides T203 [14]. Here we show that the same mutants are not able to afford tolerance, both to cucumber and Arabidopsis, during germination under salt stress imposition ( Figure 6). This suggest that Trichoderma fungi, similarly to PGPR bacteria, can ameliorate plant growth under abiotic stressful conditions by lowering deleterious elevated ethylene levels accompanied by an elevated antioxidative capacity.
As mentioned above, Trichoderma stimulate biomass production and lateral root growth promotion by production of auxin [39]. This is supported by induction upon Trichoderma colonization of genes such as ASA1 and MYB77 ( Figure 1A and 1B). In the root, ethylene and auxin can reciprocally regulate each other's biosyntheses [60,61] proposed that Trichoderma IAA contributes to exogenous auxin-stimulated ethylene biosynthesis via ACC synthase. In this model, Trichoderma ACCD activity reduces the availability of ACC necessary for ethylene biosynthesis and the reductions in ethylene promote plant growth via gibberellins (GA) signaling by increasing the degradation of DELLA proteins that are repressors of gibberellin (GA) signaling. Moreover, gibberellins may control the onset of JA-and SA-dependent defense responses of the plant through the regulation of DELLA protein degradation [60]. It thus appears that defense occurs at the expense of growth. Supporting this view, recent studies have uncovered new roles for both JAZ and DELLA proteins in the regulation of JA-GA crosstalk as well as the conflicting association between defense and growth. DELLAs' positive effect on JA signaling seems to be exerted at the level of JAZ repressors as DELLAs interact with JAZ proteins and hinder their ability to repress MYC2 [62,63]. Since MYC2 has not a significant change in our present work ( Figure 4) it seems that during root colonization, growth is promoted through GA-mediated degradation of DELLAs while defense is repressed through JAZs repressing MYCs. This tips the balance towards growth, while allowing root colonization by Trichoderma.

Fungal and plant growth conditions
For microarray and gene expression analysis by qPCR, Arabidopsis Col-0, wrky18/wrky40, WRKY40-over-expressingline and the fmo1 mutant plants were grown 25 days under a long day regime (16 hours light), on rock-wool placed over a 5 L tank supplemented with Arabidopsis hydroponic solution [19]. Trichoderma inoculum was added to the root system, resulting in a concentration of 10 5 germinated spores mL 21 . Roots were collected at 9, 24 and 48 hpi. For salt stress imposition in Arabidopsis, NaCl was added to the growth medium at a final concentration of 100 mM, two days after Trichoderma root inoculation and roots were collected after two more days. Cucumber seedlings were grown in hydroponics boxes as described in [4]. For cucumber salt stress imposition NaCl was added at a final concentration of 100 mM two days after Trichoderma root inoculation.
For greenhouse experiments, cucumber or Arabidopsis seeds were planted in soil in 250 ml boxes. Trichoderma strains were mixed with the soil at a final concentration of 10 6 spores/g soil. Seeds were watered with NaCl solutions at concentrations described in each experiment, or water (control) starting from seeding. Seedlings germination was evaluated 7 days from planting. Each treatment included 4 boxes with 10 cucumber seeds or 20 Arabidopsis seeds. The experiment was repeated three independent times.

RNA extraction and microarray analysis
For microarrays analysis total RNA was purified using the RNeasy Mini Kit (Qiagen) from Arabidopsis roots pooled from 60 plants (24 hpi and from non-inoculated control plants) from two independent experiments. DNaseI digestion was performed with the Turbo DNA-free Kit (Ambion). cDNA was prepared and hybridized to Agilent Arabidopsis (V4) Gene Expression Microarrays (4644K) in the Microarrays Unit of the Weizmann Institute of Science (Rehovot, Israel), according to manufacturer instructions. The microarray data have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (Accession no. GSE42113). Statistical analysis, normalization and fold-change values of microarray data were performed using the ROBIN software [65]. The MapMan software was used in order to create MapMan overview diagrams of the microarray data [34]. Twentytwo genes (list of primers in File S5) were used to validate the microarray analysis by qPCR as described in the following section.

Expression profiling by qPCR
Total RNA was prepared as described in the previous section from roots of 35 plants. Four micrograms of total RNA were used as template for first-strand cDNA synthesis with RevertAid cDNA Synthesis Kit (Thermo Fisher Scientific). cDNA (20 ng) was used for qPCR with Power SYBR Green reagent performed on a ABI PRISM 7900HT sequence detection system (Applied Biosystems). Data were analyzed with the 7900 V2.0.3 evaluation software (Applied Biosystems).
All genes and primers used in this paper are listed in file S4. Arabidopsis primers sequences were designed using the QuantPrime online tool (www.quantprime.de) and available through the Mueller-Roeber Lab webpage. The 137 genes of the Arabidopsis expression platform have been selected by the criteria of being experimentally proven to be involved in defense response processes against biotic and abiotic stress. Cucumber primers were designed using Primer Express (Applied Biosystems). The fold change in the target genes was normalized to ACTIN2 and GADPH reference genes for Arabidopsis and the 18S reference gene for cucumber. Gene fold expression relative to control plants was determined using the DDCT as described in [66]. Three biological experiments (with two independent replicates, obtain from different roots pool, for each experiment) were performed for each treatment.

Quantification of Trichoderma root colonization by qPCR and root colonization assay
A time-course experiment was performed with inoculated Arabidopsis roots detached from 35 plants from each hydroponic growth container at 24, 48, 72, 96 and 120 hpi extensively washed in water. Plant and Fungal growth conditions as described above. After sterilization in 1% (v/v) NaOCL for 1 min, the roots were washed with sterile distilled water, and total DNA extraction was performed according to [67]. In planta quantification of T. asperelloides was followed by amplification of 200-bp and 201-bp using gene specific primers for the Trichoderma genes TasSwo [68] and b-tubulin, respectively. Primers sequences can be found in file S4. Arabidopsis ACTIN2 and GADPH genes) were used as control references for quantitative analysis. qPCR was carried out as mentioned above. Each sample was examined in triplicates using a relative quantification analysis by the standard curve technique as described by [35,69]. Root colonization assays were performed according to [70]. Briefly, roots were detached 12 h postinoculation and extensively washed in water. After sterilization in 1% NaOCl for 2 min, the roots were washed with sterile distilled water, weighed, and homogenized using an ULTRA-TURRAX apparatus (Janke & Kunkel) in 20 mL of water for 1 min. Serial dilutions were plated for colony forming unit counts on Trichoderma selective medium [71] at 28uC.

LC-MS analysis of indole glucosinolates metabolites
Secondary metabolite analysis by LC-MS was performed as described by [72]. All data were processed using Xcalibur 2.1 software (Thermo Fisher Scientific, Waltham, USA). Indole glucosinolates identification and annotation were performed using comparison with our previous publications [73,74].

Ascorbic acid determination
Measurement of reduced, oxidized and total ascorbate content in plants was done according [75] using the a-a9-bipyridyl method. Cotyledons were sampled from seedlings grown in soil with or without Trichoderma (C and +T) with or without salt stress application (+NaCl and +NaCl+T). Plant material was pooled from three different plants, ground, and 200 mg were taken in the assay.

Statistical analysis
For seedlings germination assay and ascorbic acid determination JMP7 software (SAS Institute Inc.) was used for statistical analyses. Data were analyzed using one-way ANOVA and mean comparisons were made using the Tukey-Kramer honestly significant difference multiple range test at (p,0.05). For qPCR analysis genes expression data were analyzed with one-side, unpaired Welch's t-test (p,0.05) in Excel. For microarrays analysis Welch's t-test was applied to identify differentially expressed genes. Gene lists were created by filtering the genes based on fold change and signal above background in at least one microarray. Upregulated genes were defined as those having a greater than or at least two-fold linear intensity ratio. For indole glucosinolates determination data were analyzed with one-side, unpaired Welch's t-test (p,0.001) in Excel.  Figure S2 Targeted LC-IT/ESIMS based quantification of indole glucosinolates in Arabidopsis roots 24 hours after colonization by T. asperelloides. Abbreviation: 4MI3MG, 4-methoxy-indol-3-ylmethylglucosinolate; 1MI3MG, 1-methoxy-3-indolyl-methyl glucosinolate; I3M, Indolyl-methyl glucosinolate. Each of the glucosinolate shows a significant difference (P,0.001) between the control and T. asperelloides treatment. Results are averages (6 standard deviation) of six replicates from two independent biological repetitions. Each repetition was a pool of 35 plants. (TIF) File S1 List of genes and fold changes that are significantly differentially expressed in Arabidopsis roots 24 hours after application of Trichoderma as monitored by microarray analysis.