- Top of page
- Materials and Methods
- Supporting Information
In plants, sucrose (Suc) is produced as one of the final products of photosynthesis and is the main form of photoassimilates that are transported to heterotrophic tissues, such as roots (Dennis & Blakeley, 2000; Koch, 2004). Suc movement within the plant is facilitated by well-characterized Suc/H+ symporters (Suc transporters) that move Suc against a concentration gradient (Shakya & Sturm, 1998; Noiraud et al., 2000; Barth et al., 2003; Flementakis et al., 2003; Yao Li et al., 2003; Dimou et al., 2005; Krügel et al., 2008). Expression experiments have demonstrated that Suc transporters are expressed in various tissues, including the outer cell layers of the root tips (Barth et al., 2003). As a result, expression of these transporters in the outermost root cells is a phenomenon that is probably controlled by plant cells to provide a resource for the development of heterotrophic organisms in the rhizosphere.
Plant-produced nutrients that are secreted into the rhizosphere become part of an intricate chemical communication that initiates interactions between the host and colonizing nonpathogenic microorganisms (Bais et al., 2006). As part of such a dialogue, many molecules produced by both partners are active participants in metabolite trafficking that establishes and regulates the symbiotic associations (Bais et al., 2004, 2006; Pozo et al., 2005). During the interaction between plants and mycorrhizal fungi, carbohydrate metabolism within the root cells has been demonstrated to play an essential role in the establishment and maintenance of the symbiotic association (Blee & Anderson, 1998; Nehls et al., 2001; Nehls, 2008). Plant-produced enzymes have been proposed to hydrolyze Suc, and the resulting monosaccharides are transported into the fungal cells (Blee & Anderson, 1998; Nehls et al., 2001). Importantly, the hexose concentration in the apoplast appears to control the fungal metabolism during the symbiosis, with the monosaccharides acting as nutrients and, at the same time, as signals that regulate gene expression in the fungal cell (Nehls et al., 2001; Schaarschmidt et al., 2007a,b; Nehls, 2008).
Despite the importance of plant-produced monosaccharides for the association of mycorrhizal fungi with plant roots, novel evidence has shown that the beneficial root-colonizing fungus Trichoderma virens is able to degrade Suc exuded by plants (Vargas et al., 2009). The disruption of Suc degradation in T. virens showed that Suc metabolism is involved in the control of fungal development, root colonization and proliferation in the rhizosphere.
The capability of intracellular Suc degradation in T. virens suggests that in its natural environment, this fungus obtains Suc from the surrounding medium (either the soil or from the roots) for further metabolism (Vargas et al., 2009). Molecular and physiological studies revealed that intracellular Suc metabolism is an important element of T. virens physiology in the rhizosphere, and its ability to colonize maize roots. However, the molecular mechanisms leading to the transport of the disaccharide remained to be discovered. Initially our knowledge of Suc transport in fungal species was limited to the characterization of an α-glucoside transporter from Schizosaccharomyces pombe, similar to Suc transporters from plants, able to transport mainly maltose and, to a lesser extent, Suc and other disaccharides (Reinders & Ward, 2001). During the preparation of this manuscript, a high-affinity Suc carrier from Ustilago maydis (UmSrt, similar to mosaccharide transporters from plants) was described as a virulence factor (Wahl et al., 2010).
In this report, we describe the identification and functional characterization of a plant-like Suc transporter from T. virens (TvSut) with high specificity toward Suc transport, and with biochemical and molecular properties similar to those described for plant Suc/H+ symporters. The presence of a functional Suc transporter in T. virens strongly suggests that this fungal species evolved a specific mechanism that enables the fungal cells to obtain and metabolize Suc from plant roots during the symbiotic association. Additionally, evidence for a regulatory network that depends on Suc metabolism and regulates several aspects of the symbiotic association is presented.
Overall, this research expands our knowledge of Suc metabolism in T. virens and discloses novel regulatory aspects during symbiotic associations. In contrast to the role of UmSrt in the interaction between maize and U. maydis, our results demonstrate that a plant-like Suc transporter in T. virens is induced during the establishment of a beneficial interaction with plants.
- Top of page
- Materials and Methods
- Supporting Information
Chemical communication and metabolite exchange play essential roles in the establishment and maintenance of symbiotic associations; in the rhizosphere, plants provide a rich environment that supports those associations and microbial communities (Bais et al., 2004, 2006; Pozo et al., 2005). Driven by the beneficial effects afforded by Trichoderma species to crops, comprehension of the molecular events that govern the association of these fungi with roots and the colonization of the rhizosphere has increased in the last decades (Harman et al., 2004). As part of the molecular events associated with the symbiotic interactions of T. virens and maize, an intracellular invertase (TvInv) is produced in the fungal cells for degradation of plant-derived Suc (Vargas et al., 2009). In this report, we demonstrate that Suc uptake into T. virens cells is mainly imported by TvSut, a specific Suc transporter with biochemical properties similar to plant-encoded Suc carriers (Lu & Bush, 1998; Shakya & Sturm, 1998; Meyer et al., 2000; Noiraud et al., 2000; Weise et al., 2000; Weschke et al., 2000; Reinders et al., 2002; Barth et al., 2003; Ramsperger-Gleixner et al., 2004; Krügel et al., 2008).
The identification and functional characterization of TvSut complements the previous study on the ability of mycelia of T. virens to metabolize Suc (Vargas et al., 2009). The results presented in this study show that during its symbiotic interaction, T. virens obtains Suc from the rhizosphere by a highly specific mechanism resembling the process that U. maydis and plants use for Suc mobilization (Wahl et al., 2010). However, TvSut is more closely related to both Suc transporters from plants and putative Suc symporters from other fungal species than to UmSrt (high-affinity Suc carrier from U. maydis) (Fig. 2, Table S3). When UmSrt was included in the phylogenetic studies, this Suc carrier did not group with any of the clades containing plant or fungal sequences (not shown). This suggests that, unlike TvSut, UmSrt1 might not belong to the well-known families of Suc transporters from plants. However, we also found two genomic regions in U. maydis encoding putative plant-like Suc transporter homologs of TvSut (Fig. 2, Table S2). An examination of the fungal sequences revealed that despite being closely related, TvSut and the functionally characterized SpSut grouped in different phylogenetic clades (Fig. 2). These observations suggest an early radiation of Suc transporters in the fungal lineage, and, in the case of T. virens, Suc-rich niches (i.e. the rhizosphere or within plant tissues) might have selected for a Suc-specific transporter with very similar properties to those of plants. The presence of putative transporters in many fungal strains, with a high degree of similarity to TvSut (Table S2), suggests that plant-like Suc transporters are probably present in other fungal species. However, their specificity, biochemical properties, and physiological role presumably will differ depending on the evolutionary history of each species and the natural environments they inhabit. In the case of the rhizosphere competent species T. virens, TvSut is related to the establishment of a beneficial association with plant roots, but in the case of a pathogenic fungus, Suc uptake is associated with virulence factors (Wahl et al., 2010).
For the optimal use of resources, a coordinated regulation of the expression of genes involved in the metabolism of such resources is required. In mycorrhizal fungi, the up-regulation of fungal hexose-transporters and enhanced carbohydrate metabolism during root colonization has been demonstrated (Blee & Anderson, 1998; Hahn & Mendgen, 2001; Nehls et al., 2001; Nehls, 2008). In the fungal species Thermomyces lanuginosus, previous studies suggested a simultaneous uptake of radiolabeled Suc and induction of invertase activity when the fungal cells were cultured in the presence of Suc (Chaudhuri et al., 1999). Similarly, the pattern of mRNA accumulation for tvsut, during root colonization or saprophytic growth, parallels the expression of tvinv encoding the T. virens intracellular invertase (Vargas et al., 2009).
The simultaneous induction of tvinv and tvsut in T. virens cells suggests a strict coordination and regulation of the mechanisms controlling gene expression in a Suc-dependent manner at early stages of root colonization (Fig. 4). One of the most intriguing issues is how the presence of Suc is perceived, resulting in the expression of tvinv and tvsut. Suc uptake experiments suggested the presence of a TvSut-independent entry of Suc into T. virens cells, as protoplasts from Δtvsut mutant strains were still able to incorporate low amounts of Suc (Figs 6b, S4). The presence of an alternative pathway for Suc uptake into the cell may be a key element for the initial Suc perception and induction of tvsut and tvinv. In addition to tvsuC, a second homolog to plant Suc transporters (denoted as TvSuC, with 50% similarity to TvSut and 30% similarity to CsSut1) was identified in the T. virens genome (accession no. FN677489, Fig. S1). However, tvsuC expression was not detected in wild-type or in Δtvsut strains cultivated in the presence of Suc or in the presence of plants (Fig. S3). Moreover, the expression of tvsuC was not detected, by Northern blotting or RT-PCR experiments, in T. virens cells cultured in the presence of glucose or glycerol (data not shown). The lack of expression of tvsuC suggests that this putative transporter is not related to Suc uptake in the conditions assayed. Sequence analyses of the T. virens genome revealed no homologous sequences to bacterial-type Suc carriers previously described, such as Suc permease or Suc-specific phosphotransferase transporters (Slee & Tanzer, 1982; Kakinuma & Unemoto, 1985; Gunasekaran et al., 1990; Postma et al., 1993, 1996; Jahreis et al., 2002; Kim et al., 2004). Additionally, no homologous sequences to UmSrt were identified in T. virens genome. Based on these observations, none of the well-known pathways for Suc uptake seem to be conserved in T. virens. However, putative unspecific disaccharide permeases, detected in the genome of T. virens (not shown), may be responsible for the initial entry of the disaccharide. This type of transporter is known to translocate a wide variety of sugars, including Suc, with different affinities in various microorganisms (Mortberg & Neujahr, 1986; Cheng & Michels, 1989; Cuneo et al., 2009). Thus, in T. virens, any of these unspecific disaccharide transporters may allow a small amount of Suc inside the cell to trigger the expression of Suc metabolism-related genes.
Previous observations suggested that the hydrolysis of plant-produced Suc inside the fungal cells is important for controlling root colonization (Vargas et al., 2009). Studies of the T. virens–maize association demonstrated that the null expression of tvsut did not affect the production of secreted hydrolytic enzymes (data not shown) or the ability of the fungal hyphae to colonize maize roots (Fig. 8c). Possibly the TvSut-independent Suc influx, discussed earlier, is sufficient to trigger the regulatory pathways leading to a controlled root colonization and hyphal growth in Δtvsut mutants. Then, T. virens cells would be able to use any alternative source of carbon from the plant to support growth inside roots. It is interesting to recall that despite being fully impaired on Suc degradation, the Δtvinv strain was still able to colonize plant roots. These phenomena suggest that when T. virens is unable to use Suc in the rizhosphere, alternative carbon sources can be utilized to support hyphal growth.
The expression of a functional tvsut is crucial for the up-regulation of tvinv, but the expression of tvsut does not require the functional expression of tvinv (Table 3). This observation suggests that Suc uptake by TvSut is a major event that enhances the expression of tvinv, which assures efficient metabolism of the disaccharide. Interestingly, the expression of cell wall-degrading enzymes (CWDEs) and the elicitor sm1 is up- and down-regulated, respectively, in mutant strains impaired in tvinv expression (Table 3, Vargas et al., 2009). We speculate that there exist at least two different Suc-dependent pathways in T. virens that can affect the regulation of gene expression; one that depends on the presence of the disaccharide in the cytoplasm (Fig. 9a), and another that is effective after its hydrolysis (Fig. 9b). Initially, an unspecific transporter (such as disaccharide permeases) would carry a small amount of Suc inside the cell to activate the expression of tvsut and tvinv (Fig. 9a). Once tvinv is expressed, a second network dependent on Suc hydrolysis is activated to control additional developmental events such as root colonization, production of CWDEs, and the elicitor Sm1 (Fig. 9b). These statements reflect our working hypothesis to explain the observed phenomena related to Suc perception and metabolism at early stages of root colonization, and the consequences on the symbiotic association in later stages. Additional experiments are being conducted to further clarify the molecular mechanisms involved in regulation of gene expression and carbohydrate metabolism inside the Trichoderma cells during rhizosphere colonization.
Figure 9. Schematic representation of the sucrose (Suc)-dependent events likely to lead to the control of the symbiotic association between Trichoderma virens and plant roots. Initially, an unspecific transporter would carry low amounts of Suc inside the cell to prime the expression of tvsut and tvinv (a). Later, once tvinv is being expressed, a second network dependent on Suc hydrolysis is activated to control additional aspects related to the establishment of the symbiotic association (b). Then the expression of tvsut will amplify the original signal by transporting increased amounts of Suc that keep the regulatory events on. CWDEs, cell wall-degrading enzymes.
Download figure to PowerPoint
The most studied models for sugar perception and signal transduction are among plants and yeasts (Rolland et al., 2006; Ramon et al., 2008). For instance, Suc signaling in plants can be mediated at two different stages: associated with the transport of the disaccharide, or after hydrolysis and metabolism (Lalonde et al., 1999; Koch, 2004; Rolland et al., 2006). In agreement with the regulatory phenomenon in plants, it is likely that T. virens acquired not only a very efficient pathway for Suc degradation similar to plants, but also inherited the mechanisms related to the perception and signaling induced by the disaccharide.