Plant-beneficial effects of Trichoderma and of its genes

Trichoderma spp. can colonize root intercellular spaces

Trichoderma strains are found in many root ecosystems. Similarly to the situation with mycorrhizae, the highly hydrated polysaccharides of the root-secreted mucigel layer and the mono- and disaccharides excreted by plant roots into the rhizosphere encourage growth of the fungi. It has been observed that plant-derived sucrose is an important resource provided to Trichoderma cells to facilitate root colonization, the coordination of defence mechanisms, and increased rate of leaf photosynthesis (Vargas et al., 2009). Solute transporters such as a di/tripeptide transporter and a permease/intracellular invertase system involved in the acquisition of root exudates have been described in Trichoderma (Vizcaíno et al., 2006; Vargas et al., 2009). Strains able to promote plant growth and provide protection against infections must be able to colonize plant roots. Colonization involves an ability to recognize and adhere to roots, penetrate the plant, and withstand toxic metabolites produced by the plant in response to invasion. In Trichoderma, adherence to the root surface can be mediated by hydrophobins, which are small hydrophobic proteins of the outermost cell wall layer that coat the fungal cell surface, and expansin-like proteins related to cell wall development. Trichoderma asperellum produces the class I hydrophobin TasHyd1, which has been shown to support the colonization of plant roots, possibly by enhancing its attachment to the root surface and protecting the hyphal tips from plant defence compounds (Viterbo & Chet, 2006), and the swollenin TasSwo, an expansin-like protein with a cellulose-binding domain able to recognize cellulose and modify the plant cell wall architecture, facilitating root colonization (Brotman et al., 2008). Plant cell-wall-degrading enzymes are also involved in active root colonization, as occurs with the endopolygalacturonase ThPG1 from Trichoderma harzianum (Morán-Diez et al., 2009).

Seventy-two hours after root colonization, Trichoderma yeast-like cells were observed together with a strengthening of plant epidermal and cortical cell walls and the deposition of newly formed barriers containing large amounts of callose and infiltrations of cellulose (Chacón et al., 2007). Typical host reactions to Trichoderma were found beyond the sites of potential fungal penetration and, unlike P. indica colonization, which does not induce plant cell wall reinforcement, callose-enriched wall appositions were apparently efficient in the restriction of fungal growth to the intercellular spaces of the epidermis and cortex, preventing the entry of Trichoderma into the vascular stele (Yedidia et al., 1999). Plants also react against fungal invasion by synthesizing and accumulating antimicrobial compounds. The ability to colonize plant roots depends strongly on the capacity of each strain to tolerate them. In Trichoderma, this resistance has been associated with the presence of ABC transport systems, which are key factors in the multiple interactions established by Trichoderma biocontrol strains with other microbes in a potentially toxic or antagonistic environment (Ruocco et al., 2009), with rapid degradation of the phenolic compounds exuded from plants (Chen et al., 2011), and with the suppression of phytoalexin production, as detected in Lotus japonicus during colonization with Trichoderma koningii (Masunaka et al., 2011). In a proteome analysis, a small secreted cysteine-rich protein (SSCP) was identified in T. harzianum and T. atroviride, proving to be a homologue of the avirulence protein Avr4 from Cladosporium fulvum (Harman et al., 2004). It has been proposed that the binding of Avr4 to chitin could protect Trichoderma against plant chitinases (Stergiopoulos & de Wit, 2009).

Induction of plant defences by Trichoderma

In addition to pre-formed physical and chemical barriers, plants have an immune system that is able to detect motifs or domains with conserved structural traits typical of entire classes of microbes but not present in their host, namely the pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively). MAMP-triggered plant responses are elicited rapidly and transiently. Early MAMP responses involve ion fluxes across the plasma membrane, the generation of reactive oxygen species (ROS), nitric oxide, ET and also, but later, the deposition of callose and the synthesis of antimicrobial compounds. Many MAMPs have been identified for PGPR, such as flagellin or lipopolysaccharides, but also secreted compounds including antibiotics, biosurfactants and volatile organic compounds have been shown to elicit systemic resistance. Effective Trichoderma strains produce a variety of MAMPs (Table 1), which to date are those most widely described among plant-beneficial fungi (Lorito et al., 2010). The first recognized Trichoderma MAMP was identified as an ET-inducing xylanase (Xyn2/Eix), produced by Trichoderma as a potent elicitor of plant defence responses in specific tobacco and tomato cultivars (Rotblat et al., 2002). The Eix epitope recognized by plants consists of five surface-exposed amino acids that are not involved in enzyme activity. Trichoderma-activated and heat-denatured cellulases also elicit melon defences through the activation of the SA and ET signalling pathways respectively (Martínez et al., 2001). Some Trichoderma proteins involved in root colonization can also act as MAMPs. Swollenin TasSwo (Brotman et al., 2008) stimulates defence responses in cucumber roots and leaves and affords local protection against fungi and bacteria, and the endopolygalacturonase ThPG1 (Morán-Diez et al., 2009) generates a response in Arabidopsis similar to the ISR triggered by PGPR. Orthologues of the SSCP cerato-platanin family – Sm1 from T. virens and Epl1 from T. atroviride – are accumulated in the hyphae during root colonization and act as MAMPs in cotton and maize (Djonović et al., 2006; Seidl et al., 2006). A glycosylation mechanism keeps these proteins in a monomeric form necessary to elicit the ISR response (Vargas et al., 2008).

Chitin oligosaccharides act as elicitors of defence responses in plants, and the scavenging of such oligomers is fundamental to the lifestyle of fungal pathogens upon colonization of their hosts (de Jonge et al., 2010). As a mechanism for perceiving chitin, plants probably developed chitinases to release the active polymers from the cell walls of invading fungi, thereby triggering defence responses. Thus, the mycotrophic activity of Trichoderma chitinases can also release chitooligosaccharides and indirectly contribute to the induction of this defence mechanism.

Certain secondary metabolites produced by Trichoderma exert an antimicrobial effect at high doses but act as MAMPs and as auxin-like compounds at low concentrations. At 1 p.p.m., 6-pentyl-α-pyrone, harzianolide and harzianopyridone activate plant defence mechanisms and regulate plant growth in pea, tomato and canola (Vinale et al., 2008), suggesting that plants’ defence mechanisms and their developmental responses to Trichoderma share common components. Peptaibols are linear peptide antibiotics of 5 to 20 amino acid residues generated by non-ribosomal peptide synthetase activity. Alamethicin, a 20mer peptaibol from T. viride, elicits JA and SA biosynthesis in lima bean (Engelberth et al., 2001), whereas 18mer peptaibols from T. virens elicit systemic defences in cucumber against the leaf pathogen Pseudomonas syringae pv. lachrymans (Viterbo et al., 2007). The protection of tobacco plants against tobacco mosaic virus by Trichoderma peptaibols was shown to involve multiple defence signalling pathways (Luo et al., 2010).

Defence signalling pathways induced by Trichoderma

Trichoderma had received little attention as a potential inducer of plant resistance until the publication of studies describing that bean root colonization by T. harzianum was effective in inducing defence responses (De Meyer et al., 1998) and that penetration of T. asperellum in the root system triggered ISR in cucumber seedlings (Yedidia et al., 1999).

As a consequence of Trichoderma root colonization and MAMP interaction the proteome and transcriptome of plant leaves are systemically affected (reviewed by Shoresh et al., 2010). The ISR triggered by Trichoderma occurs through the JA/ET signalling pathway similarly to PGPR-ISR (Shoresh et al., 2005), as confirmed by several authors: (i) cerato-platanin Sm1 is required for T. virens-mediated ISR against Colletotrichum graminicola in maize (Djonovic et al., 2007), (ii) Trichoderma treatment of JA/ET-deficient Arabidopsis genotypes leads to enhanced susceptibility to Botrytis cinerea (Korolev et al., 2008), (iii) ISR triggered by PGPR and Trichoderma converges upstream from MYB72, an early key component of the onset of ISR (Segarra et al., 2009). However, other studies have shown that in the T. asperellum–cucumber interaction the induction of plant responses is a time- and concentration-dependent phenomenon, and in the first hours of contact a SAR-like response is observed, with an increase in SA and peroxidase activity. In fact, a systemic increase in SA and JA levels was observed after inoculation of high densities of Trichoderma (Segarra et al., 2007). Gallou et al. (2009) also observed that the defence response of T. harzianum-challenged potato to Rhizoctonia solani was dependent on JA/ET and SA.

Recent findings include: (i) the colonization of Arabidopsis roots by T. atroviride induces a delayed and overlapping expression of the defence-related genes of the SA and JA/ET pathways against biotrophic and necrotrophic phytopathogens, both locally and systemically (Salas-Marina et al., 2011); (ii) Trichoderma is able to trigger a long-lasting upregulation of SA gene markers in plants unchallenged by pathogens, although when plants are infected by a pathogen such as B. cinerea, the pretreatment with Trichoderma may modulate the SA-dependent gene expression and, soon after infection, the expression of defence genes induced through the JA signal transduction pathway occurs, causing ISR to increase over time (Tucci et al., 2011); and (iii) colonization of Arabidopsis root by T. asperellum produces a clear ISR through an SA signalling cascade, and both the SA and JA/ET signalling pathways combine in the ISR triggered by cell-free culture filtrates of Trichoderma (Yoshioka et al., 2011).

Plant growth promotion and tolerance to abiotic stress

Certain Trichoderma spp. have beneficial effects on plant growth and enhance resistance to both biotic and abiotic stresses. Early work revealed that Trichoderma promotes growth responses in radish, pepper, cucumber and tomato (Baker et al., 1984; Chang et al., 1986). Further studies demonstrated that Trichoderma also increases root development and crop yield, the proliferation of secondary roots, and seedling fresh weight and foliar area (Harman, 2000). Moreover, T. harzianum can solubilize several plant nutrients (Altomare et al., 1999), and the colonization of cucumber roots by T. asperellum has been shown to enhance the availability of P and Fe to plants, with significant increases in dry weight, shoot length and leaf area (Yedidia et al., 2001).

Tucci et al. (2011) have recently demonstrated the effects of the plant genetic background on the outcome of the interaction between different tomato lines and two biocontrol strains of T. atroviride and T. harzianum, and in at least one tomato cultivar the Trichoderma treatment did not exert any plant growth promotion effect and was even seen to be detrimental. The growth-promoting activity of T. atroviride on tomato seedlings has been suggested to be associated with the reduced ET production resulting from a decrease in its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) through the microbial degradation of indole-3-acetic acid (IAA) in the rhizosphere, and/or through the ACC deaminase (ACCD) activity present in the micro-organism (Gravel et al., 2007). Putative sequences of ACCD have been found in Trichoderma genomes (Kubicek et al., 2011), and RNAi silencing of the ACCD gene from T. asperellum revealed a decreased ability of the mutants to promote the root elongation of canola seedlings, suggesting a role for ACCD in root growth promotion (Viterbo et al., 2010). Moreover, Trichoderma spp. produce auxins that are able to stimulate plant growth and root development (Contreras-Cornejo et al., 2009). As indicated above, an auxin-like effect has been observed in etiolated pea stems treated with harzianolide and 6-pentyl-α-pyrone, the major secondary metabolites produced by different Trichoderma strains (Vinale et al., 2008). Maize rhizosphere colonization by T. virens also induces higher photosynthetic rates and systemic increases in the uptake of CO₂ in leaves (Vargas et al., 2009).

The beneficial effects of Trichoderma on abiotic stress have been well documented (Donoso et al., 2008; Bae et al., 2009), although the mechanisms controlling multiple plant stress factors are still unknown. Recently, Mastouri et al. (2010) reported that the treatment of tomato seeds with T. harzianum accelerates seed germination, increases seedling vigour and ameliorates water, osmotic, salinity, chilling and heat stresses by inducing physiological protection in plants against oxidative damage. These responses are comparable with the effects induced in plants by P. indica, which shows strong growth-promoting activity during its symbiosis with a broad spectrum of plants and induces resistance to fungal diseases and tolerance to salt stress (Vadassery et al., 2009). A common mechanism through which beneficial fungi and PGPR enhance plant tolerance to these abiotic stresses could be the amelioration of damage caused by ROS accumulation in stressed plants (Mastouri et al., 2010).

Trichoderma–plant cross-talk model

Plant immunity and development are interconnected in a network of hormone-signalling pathways (Pieterse et al., 2009). There is a cross-communication between SA, JA and ET, the central players in defence, and the response pathways of other hormones such as abscisic acid, commonly associated with plant development and abiotic stress, IAA, related to plant growth and lateral root development, and gibberellins, which control plant growth by regulating the degradation of growth-repressing DELLA proteins. In Trichoderma, the ACCD activity reduces the availability of ACC necessary for ET biosynthesis (Fig. 2). Reductions in ET promote plant growth via gibberellin signalling by increasing the degradation of DELLA proteins. Moreover, gibberellins may control the onset of JA- and SA-dependent defence responses of the plant through the regulation of DELLA protein degradation. ET and IAA in the roots can reciprocally regulate each other’s biosynthesis (Stepanova et al., 2007) and, according to this observation, Trichoderma IAA contributes to exogenous auxin-stimulated ET biosynthesis via ACC synthase, which in turn triggers an increase in abscisic acid biosynthesis. Depending on the timing and outcome of Trichoderma stimuli, phytohormone homeostasis will control plant development and immune responses.

Transgenic plants expressing Trichoderma genes

The pioneering work of Lorito et al. (1998) demonstrated that Trichoderma genes can be expressed functionally in plants to confer beneficial features, mainly in the control of plant diseases. In that work, high expression levels of the T. harzianum endochitinase gene chit42 were obtained in different plant tissues, with no visible effect on plant growth and development. Selected transgenic lines of tobacco and potato were highly tolerant or completely resistant to the leaf pathogens Alternaria alternata, Alternaria solani and B. cinerea, and the soil-borne pathogen Rhizoctonia solani. chit42 expression increased resistance to Venturia inaequalis, but reduced plant growth, in apple (Bolar et al., 2000) and significantly increased the resistance of broccoli to attack by Alternaria brassicicola (Mora & Earle, 2001). The expression of chit42 in lemon enhanced resistance to Phoma tracheiphila and B. cinerea, a significant correlation between resistance and transgene expression being observed, with an upregulation of ROS and JA/ET-responsive genes (Gentile et al., 2007; Distefano et al., 2008). The homologous chit42 gene from T. virens was able to enhance resistance against R. solani when it was expressed in rice (Shah et al., 2009). Other Trichoderma chitinase genes have been used to generate transgenic plants resistant to fungal diseases: (i) tobacco cell cultures expressing the T. harzianum endochitinase chit40 gene released the enzyme into the medium and were able to inhibit the conidial germination of the post-harvest pathogen Penicillium digitatum (Brants & Earle, 2001); (ii) transgenic cotton plants expressing the T. virens endochitinase gene Tv-ech1 showed significant resistance to A. alternata and R. solani and a rapid and greater induction of ROS, followed by modulation of the expression of several defence-related genes and the induction of the terpenoid pathway (Emani et al., 2003; Kumar et al., 2009); (iii) the expression of the endochitinase chit36 gene of T. harzianum in carrot significantly enhanced tolerance to A. radicina and B. cinerea (Baranski et al., 2008).

Co-expression of endo- (ech42) and exo- (nag70) chitinases of T. atroviride in apple has been correlated with increased resistance to V. inaequalis, and a negligible reduction in vigour (Bolar et al., 2001; Faize et al., 2003). In a similar way, the multiple expression of rice transgenes encoding two chitinases (ech42 and nag70) and one β-1,3-glucanase (gluc78) of T. atroviride resulted in resistance to R. solani and Magnaporthe grisea in rice (Liu et al., 2004). The generation of innate defence responses and enhanced salt stress tolerance was observed in tobacco plants overexpressing the T. harzianum chit33 and chit42 chitinase genes (Dana et al., 2006), again opening the possibility that chitinases might act on endogenous plant fungi by releasing chitooligosaccharides as general inducers of defence responses.

Abiotic stress tolerance in plants is accompanied by growth inhibition after overexpression of heat-shock genes of plant origin (Cazalé et al., 2009). Thus, an important biotechnological advantage has been the expression of a T. harzianum hsp70 gene in Arabidopsis to induce resistance to high temperatures, high salinity and drought without loss of vigour and growth or developmental alterations (Montero-Barrientos et al., 2010), probably due to the heterologous nature of the transgene. Recent examples of biotechnological solutions from Trichoderma are the T. harzianum Thkel1 gene, encoding a kelch-repeat protein involved in the modulation of glucosidase activity that enhanced seed germination and plant tolerance to salt and osmotic stresses when it was expressed in Arabidopsis (Hermosa et al., 2011), and transgenic tobacco plants expressing a T. virens glutathione transferase to improve their remediation and xenobiotic degradation potential (Dixit et al., 2011).