The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride

Abstract

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

About the Authors:

Rossana Segreto

Contributed equally to this work with: Rossana Segreto, Hoda Bazafkan

Roles Conceptualization, Data curation, Investigation, Methodology, Validation, Visualization, Writing – original draft

Affiliation: Department of Microbiology, University of Innsbruck, Innsbruck, Austria

ORCID logo https://orcid.org/0000-0002-2566-7042

Hoda Bazafkan

Contributed equally to this work with: Rossana Segreto, Hoda Bazafkan

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft

Affiliation: Department of Microbiology, University of Innsbruck, Innsbruck, Austria

Julia Millinger

Roles Investigation

Affiliation: Department of Microbiology, University of Innsbruck, Innsbruck, Austria

Martina Schenk

Roles Investigation

Affiliation: Department of Microbiology, University of Innsbruck, Innsbruck, Austria

Lea Atanasova

Roles Data curation, Investigation, Methodology, Validation, Visualization, Writing – original draft

Affiliation: Institute of Food Technology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

ORCID logo https://orcid.org/0000-0002-1751-277X

Maria Doppler

Roles Formal analysis, Investigation

Affiliation: Department of Agrobiotechnology IFA-Tulln, Center for Analytical Chemistry, University of Natural, Resources and Life Sciences, Vienna (BOKU), Tulln, Austria

Christoph Büschl

Roles Formal analysis, Investigation, Methodology

Affiliation: Department of Agrobiotechnology IFA-Tulln, Center for Analytical Chemistry, University of Natural, Resources and Life Sciences, Vienna (BOKU), Tulln, Austria

Mélanie Boeckstaens

Roles Conceptualization, Formal analysis, Investigation, Methodology, Supervision, Validation, Visualization, Writing – original draft

Affiliation: Département de Biologie Moléculaire, Laboratory of Biology of Membrane Transport, Université Libre de Bruxelles, Gosselies, Belgium

ORCID logo https://orcid.org/0000-0003-1629-7403

Silvia Soto Diaz

Roles Investigation

Affiliation: Département de Biologie Moléculaire, Laboratory of Biology of Membrane Transport, Université Libre de Bruxelles, Gosselies, Belgium

ORCID logo https://orcid.org/0000-0002-1958-2587

Ulrike Schreiner

Roles Investigation

Affiliation: Department of Microbiology, University of Innsbruck, Innsbruck, Austria

Fabiano Sillo

Roles Data curation, Formal analysis, Investigation, Methodology, Writing – original draft

Affiliation: IPSP-CNR, Torino, Italy

ORCID logo https://orcid.org/0000-0002-1218-9985

Raffaella Balestrini

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Writing – original draft

Affiliation: IPSP-CNR, Torino, Italy

ORCID logo https://orcid.org/0000-0001-7958-7681

Rainer Schuhmacher

Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Writing – original draft

Affiliation: Department of Agrobiotechnology IFA-Tulln, Center for Analytical Chemistry, University of Natural, Resources and Life Sciences, Vienna (BOKU), Tulln, Austria

ORCID logo https://orcid.org/0000-0002-7520-4943

Susanne Zeilinger

Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

* E-mail: [email protected]

Affiliation: Department of Microbiology, University of Innsbruck, Innsbruck, Austria

ORCID logo https://orcid.org/0000-0003-3112-0948

Introduction

Several Trichoderma species are potent mycoparasites known as biocontrol agents for the protection of plants against fungal phytopathogens. Necrotrophic mycoparasites such as Trichoderma kill other fungi through the production of damaging enzymes and toxic metabolites followed by feeding on the released nutrients [1]. While the mechanisms contributing to the mycoparasitic activity are well characterized, several knowledge gaps exist about the regulatory processes and pathways involved in activating the mycoparasitic response. During recent years, evidence accumulated that G protein signaling, the cAMP pathway, and mitogen-activated protein kinase (MAPK) cascades affect growth and development as well as the mycoparasitic activity in response to environmental cues (reviewed in [1, 2]).

The target of rapamycin (TOR) pathway is a central regulator of cell growth in response to nutrient availability in eukaryotes. Higher eukaryotes possess only one evolutionary conserved TOR protein kinase, while some yeast species such as Saccharomyces cerevisiae (Ascomycota, Saccharomycotina) possess two, which are active under nitrogen sufficiency and inactivated upon nitrogen starvation or by the addition of the bacterial metabolite rapamycin [3]. In yeast, the rapamycin-triggered TOR inhibition notably causes nuclear accumulation of the GATA-type transcription factor Gln3 resulting in the expression of nitrogen catabolite-repressed genes that are required for the assimilation of alternative nitrogen sources. This leads to the production of transport proteins for nitrogenous compounds, such as the general amino acid permease Gap1p and the ammonium transport proteins Mep1p, Mep2p and Mep3p, whose activity and/or stability are moreover post-translationally regulated by the protein kinase Npr1 (nitrogen permease reactivator 1) [4–7]. Under nutrient-rich conditions, when TOR is active, Npr1 is inhibited by phosphorylation. In case of TOR inhibition by rapamycin, Npr1 becomes functional allowing the TOR pathway to rapidly adjust the permeability of nutrients to regulate cell growth in response to environmental nutrient availability [8–10].

The TOR kinase functions in two distinct protein complexes, TORC1 and TORC2 [11]. Mammalian TORC1 governs metabolism and cell growth by promoting ribosome biogenesis and protein synthesis through triggering the phosphorylation of the S6 subunit of the ribosomal complex (Rps6) and the eukaryotic translation initiation factor 4E binding protein [12]. Similarly, under preferential growth conditions, S. cerevisiae TORC1 stimulates anabolic processes leading to ribosome and protein biosynthesis by activating the S6-Sch9 signaling branch, whereas it represses the Tap42-PP2A phosphatase branch required for the activation of environmental stress response, amino acid biosynthesis, nitrogen assimilation pathways and the catabolic process of autophagy [13–17]. The functions of TORC2 are less well understood. TORC2 regulates actin polarization and cytoskeleton rearrangement in most organisms studied and, in S. cerevisiae, also cell wall integrity [18, 19]. Rapamycin is an allosteric inhibitor of mainly TORC1, while TORC2 is largely unaffected. ATP-competitive inhibitors such as Torin1 and Torin2 target the kinase activity and hence act on both TORC1 and TORC2. In addition, they may as well cause inhibition of rapamycin-resistant TORC1 functions such as observed in mammalian and Schizosaccharomyces pombe (Ascomycota, Schizosaccharomycetes) cells [20, 21].

Some, but not necessarily all, components of the TOR pathway are present in animals, fungi, plants and protozoa. Complete genome sequence analyses revealed that the pathway evolved by successive addition of extra features from a primitive energy-sensing system in the ancestral eukaryote that coupled cell growth to energy supplies [22]. TSC1 and TSC2, which form the tuberous sclerosis complex (TSC), have been studied mainly in mammalian cells where TSC regulates TORC1 activity via the small G protein Rheb (Ras homolog enriched in brain) in response to various cues including specific nutrients and growth factors [23]. In addition, TSC is thought to positively regulate mammalian TORC2 independently of Rheb which, however, is poorly understood [18]. A similar situation exists in S. pombe [24, 25], whereas S. cerevisiae lacks TSC1/2 homologs but encodes a Rheb-like protein (Rhb1) [26, 27]. Aspergillus fumigatus (Ascomycota, Eurotiomycetes) is the only filamentous fungus in which a Rheb orthologue has been functionally characterized [28]. However, similar to all other Eurotiomycetes examined so far, A. fumigatus lacks Tsc1 [27]. Transcription of the A. fumigatus rheb orthologue, rhbA, was induced in response to nitrogen starvation and the growth of the rhbA deletion mutant was impaired on medium containing poor nitrogen sources [28, 29]. In addition, the ΔrhbA mutant showed enhanced sensitivity to the TOR inhibitor rapamycin and reduced virulence in a mouse model of invasive aspergillosis [28].

In fungi, the availability and quality of nutrients influence cellular key processes and nitrogen limitation emerged as one of the cues for activating the expression of virulence-related genes in plant pathogens [30, 31]. Although TOR signaling is poorly studied in filamentous fungi, an important role of this pathway in regulating virulence functions in Fusarium graminearum, F. oxysporum, and F. fujikuroi (all Ascomycota, Sordariomycetes) became apparent [31–33]. Over the years, evidence accumulated that a similar situation may exist in the mycoparasitic fungus Trichoderma atroviride (Ascomycota, Sordariomycetes) in which the presence of a living fungal host causes stress resembling nitrogen limitation [34, 35]. Based on this evidence, we aimed to characterize the role of the hitherto unstudied TOR signaling pathway in T. atroviride. By using both chemical TOR kinase inhibition as well as genetic manipulation of selected TOR pathway components, we found that T. atroviride possesses a functional TOR pathway that governs important cellular functions including vegetative growth, ribosome biogenesis, and nitrogen utilization, as well as mycoparasitism-related functions such as mycoparasitic overgrowth and secondary metabolite production.

Results

The growth of T. atroviride is sensitive to TOR kinase inhibitors

The sensitivity to the TOR kinase inhibitor rapamycin was assayed in order to get evidence if TOR signaling governs T. atroviride growth. Cultivation of T. atroviride on PDA plates amended with rapamycin revealed inhibition of radial growth at rapamycin concentrations ≥ 10 μg/ml (11 μM). Interestingly, even at higher concentrations (60 μg/ml; 66 μM) rapamycin only inhibited up to 40% of radial growth under the conditions tested (Fig 1A; S1 Table in S1 File). On minimal media (MM) with ammonium sulfate as sole nitrogen source, rapamycin more strongly interfered with fungal growth than on PDA resulting in a slight growth reduction already at a concentration of 500 ng/ml and a ~50% reduced colony size at 10 μg/ml. However, similar to PDA, rapamycin was not able to completely shut-down fungal growth on this medium even at concentrations of up to 60 μg/ml (Fig 1A). When cultivating T. atroviride on PDA supplemented with the second generation TOR inhibitor torin1, a clear dose-dependent effect on fungal growth (~25 to 80% growth inhibition in the range of 0.6 μg/ml (1 μM)– 6 μg/ml (10 μM) torin1) was observed. Higher concentrations of up to 24 μg/ml (40 μM) did not significantly inhibit growth further (Fig 1B; S1 Table in S1 File). A similar effect emerged on MM. Only combined treatment of the fungus with 30 μg/ml rapamycin and 6 μg/ml torin1 led to a nearly complete (> 90%) growth inhibition on MM (Fig 1C; S1 Table in S1 File). Altogether, the observed effects of the TOR kinase inhibitors suggest the presence of a functional TOR kinase signaling pathway that impacts vegetative growth of T. atroviride.

[Figure omitted. See PDF.]

Fig 1. Radial growth of T. atroviride in the presence of TOR kinase inhibitors.

The fungus was cultivated on solid potato dextrose agar (PDA) or minimal medium with 50 mM ammonium sulfate (AS) supplemented with different concentrations of (A) rapamycin, (B) torin1, and (C) different combinations of rapamycin (rap) and torin1 (tor). TOR kinase inhibitors were dissolved in DMSO and respective amounts of DMSO were used as control. Plates were incubated at 25°C for 48 hours in the case of PDA and for 72 hours in the case of minimal medium with 50 mM AS.

https://doi.org/10.1371/journal.pone.0262180.g001

T. atroviride possesses TORC1 and TORC2 as well as TSC complex components

Based on the evidence of a functional TOR kinase in T. atroviride, we next aimed to identify and functionally characterize selected components of the TOR pathway in this mycoparasitic fungus. BLASTp analysis of the T. atroviride genome database with S. cerevisiae Tor1p and Tor2p [36] revealed a single TOR orthologue (Ta_293155; TOR1) as previously described [37] (S2 Table in S1 File). The predicted T. atroviride TOR1 protein contains the typical conserved TOR kinase architecture [38] with nine N-terminal HEAT repeats, FAT and FATC domains, a FKBP12-rapamycin binding domain, and the phosphoinositide 3-kinase catalytic domain. Genome analysis also revealed the presence of a rapamycin-binding FKBP12 orthologue (Ta_297179) in T. atroviride, which is implicated in forming a complex with rapamycin that negatively affects TORC1 activity [39]. Furthermore, orthologues of the yeast TORC1 component Kog1 (Ta_223683), of the TORC2 components Avo1 (Ta_40578), Avo2 (Ta_239629), and Avo3 (Ta_145571) and of Lst8 (Ta_283530), which is an essential component of both TORC1 and TORC2 [40], were found. These data suggest the opportunity for T. atroviride TOR1 to form both TORC1 and TORC2 complexes.

The Ras-like small GTPase Rheb is one of the major regulators of TORC1 activity in mammalian cells [41]. A single Rheb orthologue, Ta_129536, which we named RHE2, is encoded in the T. atroviride genome [37] (S2 Table in S1 File). The activity of Rheb is regulated by the tuberous sclerosis complex (TSC), with TSC2 inhibiting TOR signaling by acting as GTPase activator for Rheb [23, 41]. The respective T. atroviride orthologues of TSC1 and TSC2 were identified as Ta_294527 (TSC1) and Ta_288880 (TSC2). The predicted T. atroviride TSC1 protein contains a harmatin domain, while the predicted T. atroviride TSC2 protein harbors a RAP GTPase-activating domain in its C-terminal part being typical for mammalian tuberin [42]. The obtained in silico evidence hence indicates the presence of a TSC complex in T. atroviride, which, based on the conserved GTPase-activating domain in Ta_288880 (TSC2), is supposed to down-regulate RHE2.

Even though a role upstream of TORC1 has been recently suggested for the fungus-specific serine/threonine kinase Npr1, it is overall known as a downstream TORC1 target in S. cerevisiae, where it is notably involved in the regulation of plasma membrane transporters such as Gap1p and Mep2p [4, 6, 8]. Recently, Pfannmüller et al. [43] reported on three genes encoding Npr1 orthologues in F. fujikuroi of which FfNPR1-1 could partially restore the growth defects of the S. cerevisiae npr1Δ mutant on ammonium, arginine and urea. Querying the T. atroviride genome database with S. cerevisiae Npr1p led to Ta_1703 as the by far most significant hit (S2 Table in S1 File). Ta_1703 has 47% amino acid (aa) identities with Npr1p and as well resulted as the best hit when using F. fujikuroi FfNpr1-1 as query (69% aa identities). With F. fujikuroi FfNpr1-2 as query, Ta_155476 emerged as the best hit (89% aa identities) while FfNpr1-2 only showed moderate (39%) amino acid identities to Ta_1703. The third Npr1 homologue of F. fujikuroi, FfNpr1-3, resulted in the identification of an additional T. atroviride serine/threonine-specific protein kinase (Ta_294017) as the best hit (68% aa identities) followed by Ta_1703 (41% aa identities) and Ta_155476 (38% aa identities). To assess if Ta_1703 (in the following named NPR1-1), which showed the highest similarity to yeast ScNpr1p, is its functional homologue, the full-length cDNA was expressed in S. cerevisiae npr1Δ mutant cells and yeast growth was tested in the presence of selected nitrogen sources [8]. Growth of yeast npr1Δ cells is reduced in the presence of several nitrogen sources, as Npr1 is essential to maintain the activity of different transporters of nitrogenous compounds [5]. As positive control of the growth test, the npr1Δ strain expressing S. cerevisiae Npr1 was used, while npr1Δ cells transformed with the empty vector served as negative control. Similar to F. fujikuroi FfNpr1-1, T. atroviride NPR1-1 complemented the growth defect of yeast npr1Δ cells slightly in the presence of urea and more efficiently in the presence of a low ammonium concentration, but not on the other nitrogen sources tested (Fig 2). These results support the hypothesis established by Pfannmüller et al. [43] that the functions of yeast Npr1 are split among different kinases in F. fujikuroi, and possibly also in T. atroviride.

[Figure omitted. See PDF.]

Fig 2. Complementation tests of S. cerevisiae npr1Δ cells with T. atroviride NPR1 in the presence of several nitrogen sources.

S. cerevisiae wild type (Wt; ura3, 23344c) and npr1Δ (npr1Δ ura3, PVV357) cells were transformed with the empty plasmid YEplac195 (-), YCpScNpr1 [8] or with YEplac195-TaNpr1p. Cells were grown in minimal buffered (pH 6.1) medium containing 3% glucose as the carbon source and glutamate (glt) 0.1%, ammonium (am) 1 mM, proline (pro) 0.1%, urea 0.1% or 1mM, citrulline (cit) 0.05% or tryptophan (trp) 0.05% as the nitrogen sources. Plates were incubated for 3, 4 or 6 days at 29°C.

https://doi.org/10.1371/journal.pone.0262180.g002

Interfering with the TOR pathway affects growth and nitrogen source utilization of T. atroviride

For functional characterization of TOR pathway components in T. atroviride, we selected tor1, tsc1, tsc2, rhe2 and npr1-1 for generating deletion mutants. At least 50 hygromycin-resistant colonies were obtained for each gene. Purification to mitotic stability accompanied by PCR-based screening resulted in six Δtsc1, four Δtsc2, three Δrhe2, and 12 Δnpr1-1 independent mutants with homologous integration of the deletion cassette at the respective target locus. After having determined the copy number of selected independent strains of the different mutants (S1 Fig) and shown that the strains carrying the same deletion behave similar in growth assays (S2 and S3 Figs), one strain of each mutant was used for further experiments. Attempts to delete the tor1 gene failed, as the generation of homokaryotic deletion mutants from the obtained hygromycin-resistant colonies that emerged from different rounds of transformation was unsuccessful. Based on these results and previous reports on the lethality of a full disruption of TOR kinase activity in several organisms including fungi [32, 33, 44], we assume that tor1 is also an essential gene in T. atroviride.

Because the TOR pathway is an important regulator of growth, we investigated the effect of the deletion of the selected TOR pathway components on T. atroviride biomass formation and radial colony growth. Biomass production in liquid complete media (PDB) was decreased in Δtsc1 and Δtsc2 mutants (by ~25% respectively), whereas Δrhe2 and Δnpr1-1 mutants grew similarly as the wild type (S3 Table in S1 File). Upon cultivation on solid complete media (PDA), a similar picture emerged (Fig 3). The Δtsc2 mutant showed decreased radial growth; Δtsc1, however, grew similar to the wild type, while Δnpr1-1 and Δrhe2 mutants showed slightly increased growth. Sporulation was comparable to the wild type in Δnpr1-1, Δrhe2 and Δtsc1 mutants, whereas Δtsc2 showed a delay in conidia formation (Fig 3, S2 Fig). The growth behavior of Δrhe2 and Δnpr1-1 mutants on MM supplemented with ammonium sulfate, arginine, or urea was similar to the wild type, while they showed slightly reduced growth on the poor nitrogen sources sodium nitrate and proline. Δtsc1 and especially Δtsc2 exhibited reduced radial growth on all of the tested nitrogen sources, in particular on nitrate. Interestingly, in the presence of glutamine as sole nitrogen source, all mutants exhibited similar colony sizes as the wild type (Fig 3; S3 Fig).

[Figure omitted. See PDF.]

Fig 3. Effect of npr1, rhe2, tsc1 and tsc2 gene deletion on mycelial growth of T. atroviride on different nitrogen sources.

Fungi were grown on PDA or on minimal medium amended with different nitrogen sources (10 or 50 mM ammonium sulfate, 10 mM L-glutamine, 10 mM L-arginine, 10 mM sodium nitrate, 10 mM L-proline, or 10 mM urea) at 25°C for three days. Growth on PDA after two and three days is shown.

https://doi.org/10.1371/journal.pone.0262180.g003

To get a more global insight into putative alterations in nitrogen utilization resulting from deletion of TOR pathway genes, we performed a comparative phenotypic profiling of the mutants and the wild type on 95 different nitrogenous compounds using the Biolog Phenotype Microarrays. Cluster analysis of the wild type nitrogen source utilization at different time points (24, 48, 72 hours) revealed four distinct clusters and several groups (S4 Fig). Such sister group of clusters I and II contained the dipeptides Gly-Met and Met-Ala which supported the best growth of T. atroviride. Cluster I also comprised nitrogen sources which yielded high mycelial density. It included mainly L-amino acids such as L-glutamine and L-asparagine, dipeptides, and urea, γ-amino-N-butyric acid and N-acetyl-D-glucosamine, the latter having been described as a preferred carbon source for T. atroviride as well [45]. Cluster II contained nitrogen sources promoting slower growth of the fungus than those in cluster I. It contained dipeptides and the amino acid L-proline but also ammonium, nitrate, and allantoin, which allowed considerable growth of T. atroviride. Cluster III nitrogen sources comprised several L- and D-amino acids that were less efficiently catabolized and which promoted only weak growth. Cluster IV nitrogen sources, on which T. atroviride only marginally grew or was unable to grow, included several amines, the amino sugars N-acetyl-D-mannosamine and N-acetyl-D-galactosamine. Pyrimidines and pyrimidine-derivatives thymine, uracil, thymidine, uridine, and alloxan were also present in cluster IV.

The nitrogen utilization profiles of the TOR pathway mutants differed from the wild type but also amongst each other. The mycelial growth of the Δtsc2 mutant was lower on most of the tested nitrogen sources compared to other strains, including ammonium, L-glutamine, and nitrate (Fig 4). Clustering based on the overall nitrogen utilization patterns of all tested strains after 72 hours of growth revealed a Δtsc1 and Δtsc2 subcluster suggesting that TSC1 and TSC2 perform similar but specific tasks compared to the other TOR pathway proteins tested (Fig 4). The Δnpr1-1 and Δrhe2 mutants had similar utilization patterns as the wild type. However, Δnpr1-1 showed enhanced growth in respect to the other tested strains on nitrite, and, together with Δrhe2, on some amino acids such as L-threonine, L-leucine, and L-methionine. Δnpr1-1 showed reduced growth on ornithine and the dipeptide Ala-Gly and for both, Δnpr1-1 and Δrhe2, a reduced growth compared to the wild type on xanthine and xanthosine was evident, which, however, was similar to Δtsc1 and Δtsc2 mutants (Fig 4).

[Figure omitted. See PDF.]

Fig 4. Comparative nitrogen source utilization profiles of T. atroviride Δnpr1, Δrhe2, Δtsc1 and Δtsc2 mutants and the wild type.

Hierarchical clustering of strains cultivated on 95 nitrogen sources and a control using the Biolog Phenotype PM3b nitrogen Microarray system at 25°C for 72 hours. Clustering was done using HCE 3.5 tool with Average Linkage (UPGMA) method implementing Euclidean Distance measure. Heat map represents the mycelial density measured at 600 nm using Tecan Sunrise plate reader with Magellan Tracker software. The color heat map from black to bright green codes for optical density ranging from 0.1 to 0.85.

https://doi.org/10.1371/journal.pone.0262180.g004

Taken together, these data reveal that the loss of TSC2 most drastically affects nitrogen source utilization and vegetative growth of T. atroviride. Although deletion of npr1 or rhe2 had no or only a minor effect on radial growth on the nitrogen sources tested in plate assays, nitrogen profiling revealed distinct effects of NPR1-1 and RHE2 on the ability of the fungus to assimilate nitrogenous compounds. In addition, clustered nitrogen utilization profiles of Δtsc1 and Δtsc2 suggest that TSC1 and TSC2 may function as a complex in T. atroviride, which is in accordance with mammalian and S. pombe TSC orthologues [24, 42]. In summary, it is evident that the nitrogen source utilization and growth of T. atroviride is TOR pathway dependent.

The TOR pathway impacts the mycoparasitic activity of T. atroviride

To assess the role of TOR signaling in T. atroviride mycoparasitism, the antagonistic behavior of the deletion mutants against Rhizoctonia solani (Basidiomycota, Agaricomycetes) as fungal host was monitored in plate confrontation assays on PDA (Fig 5) and on MM with ammonium sulfate (S5 Fig), as these conditions supported growth of both interaction partners. The soil- borne phytopathogen R. solani, that causes diseases in a wide range of crops, was selected as host due to its agricultural importance. During the early phases of the interaction, in which the mycoparasite grew towards the host and established direct contact, Δnpr1-1, Δrhe2 and Δtsc1 mutants behaved similar as the wild type on both media tested. In contrast, interaction of the Δtsc2 strain with the host fungus was delayed. After 7 days, the wild type, Δnpr1-1 and Δrhe2 had almost completely overgrown the R. solani colony while the Δtsc1 mutant showed an only incomplete overgrowth of the host fungus. Loss of tsc2 most significantly impaired mycoparasitic overgrowth as Δtsc2 mutants only unassertively attacked the host or even, upon cultivation on PDA, stopped growing at the interaction border (Fig 5). From these results, we conclude that activated TOR1 resulting from loss of TSC1 or TSC2 negatively affects the mycoparasitic overgrowth activity of T. atroviride.

[Figure omitted. See PDF.]

Fig 5. Confrontation assays for assessing the mycoparasitic activity of T. atroviride Δnpr1, Δrhe2, Δtsc1 and Δtsc2 mutants and the wild type.

Fungi were inoculated on opposite sides of a PDA plate (left side: T. atroviride; right side: R. solani) and grown at 25°C for 7 days. Pictures were taken after 3 and 7 days.

https://doi.org/10.1371/journal.pone.0262180.g005

TOR pathway interference affects T. atroviride secondary metabolite production

T. atroviride is a prolific producer of secondary metabolites, some of which have antifungal properties and hence are relevant for the mycoparasitic interaction [46]. Since TOR is known to act not only as nutrient sensitive regulator of cell growth but also as a player in nitrogen regulation of fungal secondary metabolism [32], we sought to investigate the effect of TOR signaling on the formation of secreted, nitrogen containing low-molecular weight compounds. To this end, the metabolite profiles of the mutants Δrhe2, Δtsc1, and Δtsc2, that lack TOR pathway regulatory proteins, were compared with those of wild type cultures using an isotope-assisted untargeted metabolomics approach based on liquid chromatography-high resolution mass spectrometry (LC-HRMS/MS). Cultivation of the tested strains on native and uniformly 13C-labeled glucose in the presence of native or (15NH4)2SO4 as a nitrogen source enabled the assignment of both the global and 15N-containing submetabolomes similar to [47, 48]. LC-HRMS measurements and subsequent data evaluation with MetExtract II resulted in a total of 2529 metabolite ions corresponding to 1153 fungal metabolites. Among those, a total of 364 metabolites (represented by 767 features) were found to constitute the N-containing submetabolome (S6 Fig). Detected metabolites were further characterized by their m/z values as well as their total number of carbon and nitrogen atoms.

When the data matrix consisting of 24 samples (6 replicates per strain and 338 N-containing metabolites; outliers removed) was subject to principal component analysis, three distinct clusters of samples confirmed major differences among the metabolite profiles of the tested mutants Δrhe2, Δtsc1 and Δtsc2. While Δrhe2 and Δtsc1 clearly differed from each other and the wild type (Fig 6A), no clear separation between the Δtsc2 mutant and the wild type was evident in the PCA score plot pointing to similar metabolite profiles and hence an only minor role of TSC2 in the regulation of T. atroviride secondary metabolism under the conditions tested. For a more detailed evaluation of the clustering of the investigated fungal samples, metabolite levels were compared pairwise between each of the respective mutants and the wild type (Fig 6B). In cultures of the Δrhe2 mutant, a total of 38 metabolites were altered significantly or highly significantly. Of those, 20 metabolites were more abundant in the wild type, while 18 were detected at higher levels in the mutant. In case of Δtsc1, the most pronounced differences were found between the mutant and the wild type. Of the 187 differing compounds, the large majority (n = 183) was produced in significant (n = 29) or highly significant (n = 154) lower amounts by the mutant compared to the wild type. For only four metabolites, higher levels were found in the mutant. In contrast, a comparison of the Δtsc2 mutant and the wild type revealed only one single compound to be significantly differing between the two strains. The data suggest a mainly stimulatory effect on the production of secreted N-containing low-molecular weight metabolites exerted by TSC1 in a TSC2-independent manner. RHE2 both positively and negatively governed secondary metabolite production, which altogether suggests the TOR pathway as a key player in the regulation of T. atroviride secondary metabolism.

[Figure omitted. See PDF.]

Fig 6. Profiles of low molecular weight substances secreted by T. atroviride Δrhe2, Δtsc1 and Δtsc2 mutants and the wild type.

Fungal strains were grown in six replicates in minimal medium containing either unlabeled or uniformly labeled (NH4)2SO4 and glucose as nitrogen- and sole carbon source respectively. Culture supernatants were harvested after 14 days of growth at 25°C, analyzed and full scan LC-HRMS chromatograms were processed and filtered for the N-containing submetabolome. (A) Principal component analysis (PCA) scores plot. Clustering of fungal culture samples along PC 1 and PC2 is shown based on the metabolite profiles with 338 N-containing compounds. Ellipses depict the 95% confidence intervals. While samples of the Δtsc1 and Δrhe2 mutant are clearly separated from each other and the wild type, the Δtsc2 mutant samples largely overlap with those of the wild type. (B) Volcano plots illustrating pairwise univariate comparisons of the three mutant strains Δrhe2 (left), Δtsc1 (middle) and Δtsc2 (right) versus the T. atroviride wild type based on their N-containing submetabolomes. Red dots indicate significantly (p<0.05, FC≥2), those in yellow indicate highly significantly (p<0.00015, FC≥2) differing metabolite abundances, while grey dots indicate not significantly different metabolites.

https://doi.org/10.1371/journal.pone.0262180.g006

Deletion of rhe2 renders T. atroviride hypersensitive to rapamycin and results in reduced RPS6 phosphorylation upon rapamycin treatment

We next analyzed the deletion mutants for putative alterations in their rapamycin susceptibility. While growth of Δnpr1, Δtsc1 and Δtsc2 mutants was affected by rapamycin similarly as in the wild type, the Δrhe2 mutant showed hypersensitivity as indicated by the inability to form colonies when exposed to the drug (Fig 7A).

[Figure omitted. See PDF.]

Fig 7. Effect of rapamycin on growth of T. atroviride Δnpr1, Δrhe2, Δtsc1 and Δtsc2 mutants and phosphorylation of ribosomal protein S6.

(A) Growth of T. atroviride wild type and Δnpr1, Δrhe2, Δtsc1 and Δtsc2 mutants on minimal medium with 50 mM ammonium sulfate and 10 μg/ml rapamycin (+). Control samples (-) were amended with equal amount of the solvent DMSO. Plates were incubated at 25°C and pictures were taken after three and seven days. (B) Western blot analysis of ribosomal protein S6 (RPS6) phosphorylation in T. atroviride wild type and the rapamycin hypersensitive Δrhe2 mutant after nitrogen starvation for five hours (-N) and subsequent growth in the presence (RAP) or absence (DMSO) of 20 μg/ml rapamycin in minimal media with 50 mM ammonium sulfate for two hours (three biological replicates for both rapamycin treatment and DMSO control are shown). Samples were probed for P-RPS6, total RPS6, and the loading control histone H3 using antibodies anti-phospho-(Ser/Thr) Akt substrate, anti-Rps6, and anti-H3.

https://doi.org/10.1371/journal.pone.0262180.g007

To monitor the activity of T. atroviride TOR1 in response to rapamycin, we analyzed the phosphorylation state of the ribosomal protein S6 (RPS6) by Western blotting as previously described [49, 50]. Consistent with the predicted molecular weight of T. atroviride RPS6 (Ta_256368; 27.26 kDa), a ~30 kDa band corresponding to phosphorylated RPS6 was visible in samples derived from the wild type when grown in the presence of ammonium sulfate (Fig 7B). Nitrogen starvation led to a complete loss of RPS6 phosphorylation. Rapamycin treatment in the presence of ammonium sulfate resulted in reduced levels of phosphorylated RPS6. The rapamycin hypersensitive Δrhe2 mutant reacted to rapamycin with a stronger decrease in RPS6 phosphorylation than the wild type (Fig 7B), while RPS6 largely remained phosphorylated in Δtsc1 and Δtsc2 mutants upon rapamycin treatment (S7 Fig). These data reveal that, similar to yeast and mammalian cells, analysis of the RPS6 phosphorylation state allows to monitor the activity level of T. atroviride TOR1. The results further correlate with the high resistance of the T. atroviride wild type to rapamycin (indicated by the fact that a complete dephosphorylation of RPS6 in response to rapamycin could not be obtained under the conditions tested) and the enhanced rapamycin sensitivity of the Δrhe2 mutant.

The transcriptomic response of T. atroviride to rapamycin mainly involves metabolism and ribosome/translation associated processes but also mycoparasitism-relevant genes

To gain an overview of the genes and cellular processes regulated by T. atroviride TOR1, we performed RNA-seq analyses of the wild type as well as the rapamycin hypersensitive Δrhe2 mutant after treatment with 20 μg/ml rapamycin or the solvent DMSO as a negative control. More than 260 million filtered reads were obtained of which > 90% could be mapped to the T. atroviride reference genome. Transcript levels in the rapamycin-treated samples of both strains were compared to their respective DMSO controls and gene expression differences were identified.

The gene set affected by rapamycin in the wild type (comparison 1; S8A Fig) comprised 1927 candidates with differential expression (adjusted p value threshold of 0.05) of which 1047 were down-regulated and 880 were up-regulated (Fig 8A and 8B). The top ten genes with the strongest rapamycin-triggered down-regulation (based on a list ranked according to log2 fold change values) comprised a putative ferric reductase, a terpenoid synthase, a haem peroxidase, a glutamine hydrolysing pyridoxal 5’-phosphate synthase, an amino acid transporter, a putative ATPase, and two G protein-coupled receptors (GPCRs). Differentially expressed genes (DEGs) with the strongest rapamycin-triggered up-regulation coded for a pleiotropic drug resistance protein of the ABC superfamily, a glycoside hydrolase family 89 protein, a methyltransferase, an aromatic amino acid aminotransferase, a carboxypeptidase, a putative monocarboxylate transporter, and an alcohol dehydrogenase superfamily protein (S2 File). 4856 genes emerged as differentially expressed in the Δrhe2 mutant upon rapamycin treatment (comparison 2) with 2403 being up- and 2453 down-regulated (Fig 8A and 8B). The top ten DEGs with the strongest rapamycin-triggered down-regulation in the mutant coded for a hydrophobin, an aldehyde dehydrogenase, a mitochondrial ATP synthase, a long-chain-fatty-acid-CoA ligase, a protein required for ribosome biogenesis, and two predicted transporters, while those up-regulated in a rapamycin-triggered manner coded for a predicted GPCR, glycosyltransferase family 8 and 15 proteins, a multicopper oxidase, a small secreted cysteine-rich protein, two putative peptidases, as well as the eliciting plant response-like protein EPL1 (S2 File).

[Figure omitted. See PDF.]

Fig 8. Transcriptional changes due to reduction of TOR1 activity caused by rapamycin treatment as well as by rhe2 gene deletion.

Venn diagrams illustrating the number of differentially expressed genes (DEGs) showing upregulation (A) and downregulation (B) in the four different comparisons (see S8A Fig) analyzed. The list of DEGs of each comparison is provided in S3 and S4 Files. Bubble plots displaying enriched GO terms of the DEGs emerging from comparisons 1 (C) and 3 (D). The x-axis represents the z-score, the y-axis indicates the significance of the term (-log10 adjusted p-value). The area of plotted bubbles is proportional to the number of genes assigned to the GO term.

https://doi.org/10.1371/journal.pone.0262180.g008

Interestingly, clustering analysis of the DEGs emerging from the four performed comparisons (S8A Fig) revealed that comparisons 1 (WTrap − WTDMSO = wild type response to rapamycin) and 3 (Δrhe2DMSO − WTDMSO = genes differentially expressed between mutant and wild type in the absence of rapamycin) formed a subcluster (S8B Fig). These findings indicate that reduction of TOR1 activity caused by either rapamycin or by rhe2 gene deletion results in similar transcriptional changes, i.e., 264 shared DEGs (174 commonly up- and 90 commonly down-regulated) (Fig 8A and 8B; S3 and S4 Files).

GO categorization and enrichment analyses showed that the genes involved in the rapamycin response of the wild type are mainly involved in metabolism (including metabolism of nitrogen compounds, small molecules, macromolecules, heterocycles, sulfur compounds), associated to ribosome biogenesis, gene expression and translation, and related to mitochondria-located processes, all of which were down-regulated (Fig 8C). Similar metabolic, ribosome- and mitochondria-associated processes were down-regulated in comparison 3, but, in addition, genes enriched in up-regulation of e.g. vesicle-mediated transport, signaling, cellular response to stimulus, and macromolecule modification resulted from rhe2 gene deletion (Fig 8D). Among the top ten genes being down-regulated in the Δrhe2 mutant compared to the wild type under normal growth (DMSO solvent control, i.e. absence of rapamycin; comparison 3) were, among rhe2 itself, candidates encoding a glycoside hydrolase family 16 protein, two WSC domain-containing proteins, three secreted cysteine-rich proteins, a putative GPCR, and a putative polyketide synthase (S2 File). Top ten DEGs being up-regulated in the Δrhe2 mutant compared to the wild type included genes coding for a pleiotropic drug resistance protein of the ABC superfamily, an aquaporin, a polyketide synthase domain-containing protein, a glycoside hydrolase family 89 protein, and an AAA+-type ATPase (S2 File).

Taken together, transcriptome analysis showed that, similar to other eukaryotes, T. atroviride TOR1 controls the expression of genes coding for ribosomal components and is a central hub of cellular metabolism. In addition, several DEGs with known or supposed roles in mycoparasitism and secondary metabolism were among the identified TOR1 targets. The transcriptional changes in the wild type in response to rapamycin furthermore turned out to be similar to the transcriptional changes caused by the deletion of rhe2 evidencing a role of RHE2 in TOR1 activation.

Discussion

The TOR kinase pathway is the main nutrient-responsive signaling route in eukaryotes and regulates essential cellular processes such as growth, metabolism and ribosome biogenesis. TOR signaling is well studied in yeasts such as S. cerevisiae, S. pombe and C. albicans as well as in mammalian cells. In these systems, TOR is active and promotes the synthesis of new ribosomes under nutrient (especially nitrogen and amino acid) sufficiency, while TOR activity declines, thereby leading to a reduction in ribosome biogenesis and growth, when nutrients or energy are limited or upon TOR kinase inhibition by respective drugs (reviewed in [51, 52]. Despite the importance of mycoparasitic Trichoderma species as biocontrol agents in plant protection and decades of research on the mechanisms of mycoparasitism, data on how nutrient-sensing pathways affect the lifestyle and mycoparasitic activity of these fungi are scarce.

To our knowledge, we here demonstrate for the first time that the mycoparasite T. atroviride possesses a functional TOR signaling pathway that governs processes not only required for vegetative growth and metabolism, but also for mycoparasitism. In addition to the conserved TOR kinase itself, we identified additional components of the TOR complexes 1 (TORC1) and 2 (TORC2) and functionally characterized putative regulatory proteins of the TOR signaling pathway. S. cerevisiae and S. pombe are among the best studied fungal organisms regarding TOR signaling; however, they differ to most other species, including T. atroviride, by encoding two distinct TOR kinase paralogs [38]. In addition to a single TOR protein (TOR1), T. atroviride possesses orthologues of the upstream TOR kinase regulators TSC1 and TSC2 as well as the Rheb GTPase which in S. pombe and mammalian cells regulate the activity of TORC1 [53, 54]. Based on S. cerevisiae Npr1 and its three F. fujikuroi orthologues, three NPR1 candidates could be identified in the T. atroviride genome. T. atroviride NPR1-1 emerged as the protein with the highest similarity to yeast Npr1 and was able to partially complement the growth defect of a S. cerevisiae npr1Δ mutant on ammonium and urea (Fig 2), a function being comparable to F. fujikuroi FfNpr1-1 [43]. The Npr1 serine/threonine protein kinase is controlled by TORC1 and regulates the activity of permeases for nitrogenous substances in S. cerevisiae, hence is necessary for nitrogen uptake in yeast cells grown on non-preferred nitrogen sources [4–7, 55, 56]. Deletion of npr1-1 in T. atroviride resulted in only subtle phenotypic changes, such as a slightly reduced growth on agar plates in the presence of nitrate or L-proline as sole nitrogen sources (Fig 3). Similar results have previously been reported for F. fujikuroi, where FfNPR1-1, FfNPR1-2 and FfNPR1-3 deletion mutants did not show significant growth defects on the tested nitrogen sources tempting the authors to summarize that the Npr1-like kinases have no major impact on the activity of the amino acid permease Gap1 in F. fujikuroi [43].

T. atroviride showed an unexpected resistance to rapamycin, an allosteric inhibitor of mainly TORC1. Rapamycin did not entirely inhibit T. atroviride growth even at concentrations of up to 60 μg/ml (Fig 1). A similar low rapamycin sensitivity has been reported for the zygomycete Mucor circinelloides [57] and recently also for the industrial cellulase producer Trichoderma reesei Rut-C30 [58]; in S. pombe, TORC1 is as well only incompletely inhibited by rapamycin [59]. In contrast, species of the order Hypocreales such as Fusarium graminearum, F. fujikuroi, F. oxysporum and Verticillium dahliae exhibited a significantly higher rapamycin sensitivity with concentrations in the range of 5 ng– 200 ng rapamycin per ml PDA being effective in inhibiting fungal growth [31–33, 60]. Interestingly, T. atroviride was more sensitive to the ATP-competitive inhibitor Torin1 to which it responded even stronger than V. dahliae [60], although a complete shutdown of fungal growth could not be obtained under the tested conditions. The combined use of rapamycin and torin1, the latter being active also against TORC2 and able to inhibit the rapamycin-resistant TORC1 activity [20, 21], resulted in a nearly complete inhibition of T. atroviride growth though. This suggests that both TOR kinase inhibitors are needed to reduce T. atroviride TOR1 activity to a level below that needed to support growth, probably because of TORC1 being only partially sensitive to rapamycin. Interestingly, a gene encoding a pleiotropic drug resistance protein of the ABC superfamily was among the candidates with the strongest rapamycin-triggered up-regulation in the T. atroviride wild type suggesting that the high resistance of the fungus to rapamycin could be due to its efficient detoxification by transmembrane export. In fact, various Trichoderma strains are able to resist and degrade synthetic chemicals and toxins and T. atroviride strain P1 even represents a fungicide resistant isolate that is compatible with certain fungicides used in agriculture [61, 62].

In mammalian and yeast cells, TOR signaling controls the phosphorylation of ribosomal protein S6 (Rps6) and thereby activates protein synthesis [17, 63, 64]. A collaboration in regulating Rps6 phosphorylation between TOR and protein kinase A (PKA), which mainly responds to carbon sources, has been shown for Candida albicans [50]. To monitor the activity of the TOR pathway in T. atroviride and to prove the inhibitory action of rapamycin on TOR1, we used RPS6 phosphorylation as a biochemical read-out. Starvation for nitrogen led to a complete loss of RPS6 phosphorylation, while rapamycin treatment resulted in reduced levels of phosphorylated RPS6 (Fig 7). While these data are correlated to the partial sensitivity of T. atroviride TOR1 to rapamycin, at the same time they imply a rapamycin-resistant residual activity. Rps6 phosphorylation was only marginally reduced by treatment with rapamycin also in S. pombe [21] and TORC1 and TORC2 have been shown to work in synergy in the regulation of Rps6 phosphorylation in S. cerevisiae [65].

The Rheb GTPase is conserved from yeast to human and plays important roles in TOR activation. Our results show that deletion of the single Rheb orthologue-encoding gene (rhe2) in T. atroviride results in an enhanced sensitivity to rapamycin, which was not only evident in growth assays but also on the level of RPS6 phosphorylation (Fig 7), suggesting that RHE2 is associated to the TOR signaling pathway also in T. atroviride. Accordingly, RHB1 gene deletion in C. albicans led to enhanced rapamycin sensitivity [66]. The observed normal growth in the presence of rich nitrogen sources or in complete media (PDB and PDA), but the slightly reduced growth on agar plates with the poor nitrogen sources nitrate and proline resulting from loss of rhe2 in T. atroviride, is consistent with the phenotype previously described for the A. fumigatus ΔrhbA mutant [28]. Interestingly, rhbA deletion resulted in reduced virulence of A. fumigatus in a mouse model of invasive aspergillosis [28], while the T. atroviride Δrhe2 mutant was as effective as the wild type in attacking the plant pathogen R. solani (Fig 5). Considered in context with the observed impaired antagonism of the T. atroviride Δtsc1 and Δtsc2 mutants (the TSC complex is supposed to negatively regulate RHE2-TOR signaling) a picture emerged that evidences a negative regulatory role of TOR1 activity in T. atroviride mycoparasitism. Although in the case of Δtsc1, the effect on mycoparasitism could be indirect due to the reduced growth of this mutant under the conditions tested, this model is in accordance with previous studies revealing that T. atroviride faces stress from nitrogen limitation and up-regulates genes for non-preferred nitrogen source assimilation when confronted with a living host fungus [34, 35]. Further support comes from reports on S. pombe where loss of Rhb1 function and disruption of TORC1 mimic a nitrogen starvation phenotype including activation of nitrogen-starvation gene expression [67–69].

In mammalian and S. pombe cells, TSC1 and TSC2 have been shown to form the heterodimeric TSC complex that negatively regulates the activity of Rheb and TORC1 by catalyzing GTP hydrolysis on Rheb [24, 42]. This activity is derived from the GTPase-activating domain of TSC2, while TSC1 is supposed to enhance and stabilize TSC2 activity but also bind other proteins in addition to TSC2 [70–72]. In mammals, the TSC complex acts as tumor suppressor that inhibits mTORC1 to limit undesirable cell growth [73] but also physically associates and positively regulates mTORC2 in a Rheb-independent manner [42]. Accordingly, disruption of the TSC complex leads to hyperactivated TORC1 signaling in S. pombe that abandons the scavenging of nitrogen sources [24, 25]. Deletion of either tsc1 or tsc2 in T. atroviride resulted in reduced radial growth on agar plates, which was especially evident on MM with nitrate or proline as nitrogen sources and more pronounced in Δtsc2 than Δtsc1 mutants (Fig 3). Although loss of TSC2 most drastically affected growth and nitrogen source utilization among all the mutants included in this study, the nitrogen utilization profiles of Δtsc1 and Δtsc2 mutants derived from Biolog analysis clustered together. These data indicate similar roles of the TSC1 and TSC2 proteins in regulating T. atroviride nitrogen source-dependent growth, which is in accordance with their function as components of the TSC complex. Nonetheless, Δtsc1 and Δtsc2 mutants also differed, which was particularly evident in their secondary metabolite profiles. While deletion of tsc2 only marginally affected the production of secreted N-containing low molecular weight substances, evidence for a mainly stimulatory, but TSC2-independent role in secondary metabolite production emerged for TSC1 in T. atroviride. TSC2-independent functions of TSC1 as well as noncanonical, i.e. Rheb- and TOR-independent, functions of the TSC genes have been reported in multiple organisms [72].

Despite its high resistance to rapamycin, T. atroviride experienced a significant transcriptional reprogramming in response to this drug. TOR1 inhibition by rapamycin resulted in the downregulation of various metabolic processes and processes associated to ribosome biogenesis, gene expression and translation (Fig 8). The negative effect of a reduced TOR1 activity on these cellular processes was also evident in the transcriptome of the Δrhe2 mutant, supporting the role of the RHE2 GTPase as an activator of TOR1. Genes with the strongest differential regulation due to TOR1 inhibition comprised proteins involved in ribosome biogenesis and energy production, several transporters of the MFS superfamily and amino acid transporters. These findings are in accordance with studies in mammalian and yeast cells where the transcript levels of ribosomal components are repressed by rapamycin and where TOR has been implicated in the transcriptional regulation of amino acid permeases [74, 75], thereby supporting a highly conserved role of the TOR kinase signaling pathway also in T. atroviride. Gene ID246378, encoding a candidate pleiotropic drug resistance protein of the ABC superfamily, showed the highest rapamycin-trigged upregulation in the T. atroviride wild type (log2FC 14.6) in our study. This ABC transporter could be responsible for transporting rapamycin out of the cells and thereby cause the high rapamycin resistance of T. atroviride. According to the rapamycin hypersensitivity of the Δrhe2 mutant, ID246378 was upregulated by rapamycin to a considerably lesser extent (log2FC 4.1) in the mutant than in the wild type. The TOR target genes with the highest differential regulation also included candidates with a putative function in mycoparasitism. These were, for example, terpenoid and polyketide synthases involved in secondary metabolite production, virulence associated PTH11-type GPCRs, that are necessary for appressorium formation in the plant pathogen Magnaporthe oryzae [76], a putative ferric reductase that in Crypotococcus neoformans is involved in iron acquisition and virulence [77], several peptidases such as serine carboxypeptidases and metallopeptidases [78], glycoside hydrolases of families GH15, GH 16, and GH 89 including N-acetylglucosaminidase [79], as well as small secreted cysteine-rich proteins including eliciting plant response protein EPL1 that acts as an elicitor for the induction of plant defense responses against pathogens [80, 81]. The expression of genes for glycoside hydrolases involved in plant cell wall degradation is affected by rapamycin in the plant pathogen V. dahliae [60] and the cellulase producer T. reesei [58], which is similar to our findings and evidences a role of TOR1 in the regulation of carbohydrate active enzyme expression.

To our knowledge, this is the first comprehensive study of TOR kinase signaling in a mycoparasitic fungus. Biochemical inhibition of TOR1 as well as a reverse genetics approach for elucidating the functions of TOR1 regulatory proteins revealed that the TOR signaling pathway plays a critical role in regulating growth by targeting genes being similar to those in yeast model organisms and mammals. In addition, several mycoparasitism-relevant genes and processes turned out to be under control of TOR1, which enhances our knowledge on the regulation of the mycoparasitic activity of T. atroviride. A detailed understanding of the signaling pathways that govern the responses of T. atroviride to environmental cues, including those derived from host fungi, is of utmost importance for the enhancement and targeted application of this biocontrol agent.

Materials and methods

Strains and culture conditions

T. atroviride strain P1 (ATCC 74058) was used throughout this study as the wild type and all mutants were derived from this strain. Fungal strains were cultivated at 25°C using a 12 hours light/dark cycle on either potato dextrose agar (PDA; Merck, Germany) or minimal medium (MM) [82]. For dual confrontation assays, T. atroviride was co-cultivated with the agriculturally important plant pathogen Rhizoctonia solani (pathogenic isolate obtained from the collection of the Department of Agricultural Sciences, Università degli Studi di Napoli “Federico II,” Naples, Italy) as host fungus on PDA or MM with 50 mM ammonium sulfate as sole nitrogen source. Media were supplemented with rapamycin (Selleckchem, Houston, USA; 100 mg/ml stock solution in DMSO) or Torin1 (Selleckchem; 4 mg/ml stock solution in DMSO) where indicated. For assessing radial growth rates, colony growth on agar plates was recorded twice a day for up to 10 days. For the analysis of biomass formation, 250 ml Erlenmeyer flasks containing either 50 ml of potato dextrose broth (PDB; Merck, Germany) or MM with either 10 mM ammonium sulfate, glutamine, N-acetyl-glucosamine, or proline as sole nitrogen source were inoculated with 106 fungal spores/ml medium (final concentration). After 72 hours of incubation at 25°C and 200 rpm, fungal biomass was determined by recording mycelial dry weight. Escherichia coli strains JM109 (Promega, Madison, Wisconsin) and Stellar®Top10 (Clontech, Takara Bio Europe, Saint-Germain en Laye, France) were used for plasmid construction and amplification.

Generation and verification of T. atroviride gene deletion mutants

T. atroviride gene deletion mutants were generated by transforming fungal protoplasts with linearized gene deletion cassettes containing the E. coli hph gene under control of the T. reesei gpd1 promoter and A. nidulans trpC terminator as selection marker. Deletion vectors were constructed using yeast homologous recombination as previously described [83]. To this end, gene specific primers (S5 File) were designed for PCR amplification of ~1-kb of the up- and downstream flanking non-coding regions of the respective gene of interest with overhangs to the hph resistance cassette using T. atroviride genomic DNA as template. Transformants were selected on PDA containing 200 μg/ml hygromycin B (Calbiochem, California, USA) and purified to mitotic stability by three rounds of single spore isolation. Homologous integration of the deletion constructs at the target locus and gene deletion was confirmed by PCR using one primer binding in the genomic region flanking the integration site and a second primer binding inside the hph gene (S5 File). The copy number of the deletion cassettes in the transformants was analyzed by Southern and qPCR and strains comprising one copy of the deletion cassette at the target locus were used for further experiments.

Southern blotting was performed on total genomic DNA (isolated with E.Z.N.A.® Tissue DNA Kit, Omega Bio-tek, Norcross, GA 30071, USA) digested with the restriction enzymes SacI or NheI (Thermo Scientific, Waltham, Massachusetts, USA) using DIG-labelled probes for the hph gene and following the manufacturer’s instruction (Roche, Basel, Switzerland). Hph copy number analysis of Δrhe2, Δnpr1, Δtsc1 and Δtsc2 mutants was performed by quantitative PCR as described [84] using total genomic DNA and primers HPH_F1 and HPH_R1 (S5 File). Sar1 was used as reference gene [85] and strain Δtsc1 11blC with one copy of the hph marker cassette was used for normalization. The PCR protocol consisted of an initial denaturation step (3 min at 95°C) followed by 40 cycles of denaturation (15 sec at 95°C), annealing (30 sec at 65°C) and extension (72°C for 40 sec) followed by a melting curve analysis. All samples were analyzed in three independent experiments with three replicates in each run.

Biolog phenotype array analysis

The Biolog PM3b MicroPlate assay (Biolog Inc., Hayward, CA) which comprises 95 wells with different nitrogenous compounds and a well with water as negative control was used to investigate growth rates and comparative nitrogen source utilization of the T. atroviride wild type and the TOR pathway mutants. Commercially available FF inoculation fluid (Biolog #72106) containing 16 ml of sterile ’gelling’ inoculating fluid (0.25% Phytagel and 0.03% Tween 40) was supplemented with 50 mM glucose. Conidia were collected from the tested Trichoderma strains as previously described [86] and inoculated to the test tubes to the final concentration of 2x106 spores/ml. The microplates were incubated in darkness at 25°C. Optical density at 600 nm was measured every 24 hours for up to 96 hours using a Tecan Sunrise microplate reader, which measures the turbidity and reflects biomass production on the tested substrate. For comparative analyses, OD600 values from the “72 hours” time point were used and quantitatively illustrated using the Hierarchical Clustering Explorer 3 (HCE3) [87] by applying the hierarchical clustering algorithm with average linkage and Euclidean distance measure. All measurements were performed in triplicates.

Western blotting

For Western blot analysis, fungi were cultivated in at least three biological replicates as described above. Harvested mycelia were ground in liquid nitrogen and homogenized in extraction buffer (20 mM Tris-HCl, pH 8; 110 mM KCl; 10% (v/v) glycerol; 5mM EDTA; 1 μM pepstatin A; 1 mM Pefablock SC (Sigma-Aldrich, St. Louis, USA)). After centrifugation (16,000 g, 30 min), the liquid phase was collected and the protein concentration determined according to Bradford (ROTI®Quant; Roth, Karlsruhe, Germany). ~50 μg of protein were separated by SDS-PAGE and blotted onto nitrocellulose blotting membranes (Amersham Protran, 0.2 μm, 80 x 90 mm, GE Healthcare, Chicago, Illinois, USA). After blocking with 4% BSA, membranes were incubated with the respective antibodies phospho-(Ser/Thr) Akt substrate (#9611, Cell Signaling Technology, Danvers, Massachusetts, USA), Rps6 (#ab70227, abcam, Cambridge, UK), and histone H3 used as loading control (#ab1791, abcam) followed by application of the secondary antibody Goat Anti-Rabbit IgG H&L (#ab175773, abcam). The blots were imaged using a G:box station (Syngene, Cambridge, UK). Conserved TOR-derived phosphorylation sites are present in T. atroviride RPS6 (Ta_256368; https://mycocosm.jgi.doe.gov/Triat2/Triat2.home.html; predicted molecular weight 27.26 kDa) at Ser235 and Ser236.

Metabolite profiling

T. atroviride wild type as well as Δtsc1, Δtsc2 and Δrhe2 mutants were grown on PDA plates until the cultures conidiated. From each strain, 2x103 spores/ml were inoculated in non-treated, non-coated 24-well plates (Eppendorf Cell Culture Plate) filled with 2 ml/well liquid SM minimal medium [79]. D-glucose (10 g/L) was used as sole carbon source and (NH4)2SO4 (10 mM) as sole nitrogen source. Three media were tested in parallel: unlabeled medium with native D-glucose and (14NH4)2SO4, 13C-labeled medium with U-13C glucose (Sigma Aldrich, St. Louis, Missouri, USA), and (14NH4)2SO4, and 15N-labeled medium containing native glucose and (15NH4)2SO4 (Sigma Aldrich). Each cultivation variant was prepared in six replicates. Incubation was carried out at 25°C in static semi-dark conditions. To estimate fungal growth, OD750 of the standing cultures was measured before harvesting, by using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, Massachusetts, USA). The culture supernatants were collected after 14 days, quenched with 66.6% (v/v) acetonitrile (ACN, Roth, Graz, Austria) and centrifuged at 20,000 × g for 10 min at 4°C. For both 13C-labeled and 15N-labeled supernatants, two types of pooled internal standards were prepared: to maximize metabolome coverage, the six labeled culture replicates were pooled for every strain separately and quenched by addition of ACN (1:2 (v/v) supernatant:ACN). For statistical analysis, a global pool across all strains was prepared. For metabolite detection, native supernatants were mixed 1:3 (v/v) with the respective 13C- or 15N-labeled pools resulting in a final ratio supernatant:ACN of 1:1 (v/v) while the supernatant consisted of a 1:1 (v/v) mix of native and labeled supernatants. This was carried out for every strain separately. For subsequent statistical data evaluation, a 200 μl-aliquot of each native replicate (12C-/ 14N conditions) was added to 600 μl of 13C-labeled pool. For QC purposes, 50 μl of each measurement solution were combined to give an aggregate sample used to monitor LC-HRMS and data processing performance. Negative controls were prepared for each strain by dilution of the respective 13C- or 15N-labeled pools or the native pool with the appropriate amount of H2O or acetonitrile. All samples were then analyzed with HPLC-high-resolution mass spectrometry (HRMS) in the full scan mode with the method described by [88].

For data processing, the measured LC-HRMS raw data files were converted into the mzXML format using msConvert from Proteowizard ([89]; version 3.0.19166-cc86d1f56). The files were then processed with the AllExtract module of MetExtract II [90]. In brief, the software searched for pairs of chromatographic peaks, originating from co-eluting native and 13C- or 15N-labeled metabolite ions. Both forms had to show distinct mirror-symmetric isotopolog patterns and perfectly coelute with highly similar chromatographic peak shapes. Filtered metabolite ions were aligned across all samples obtained for either the 13C- or 15N-labeled cultures. Parameter settings of MetExtract II were: Intensity threshold for M and M’: 10000 counts; Start RT: 3 minutes, End RT: 36 minutes; Wavelet min/max scale: 3; EIC extraction: ±5 ppm; Maximum intensity abundance error: ± 0.15; Minimum peak correlation: 0.85 (Pearson correlation); Maximum clustering error: max. ±8 ppm; Number of isotopologs checked: 2; Settings for the integration of the 13C- and the 15N-labeled culture-derived features: Maximum allowed m/z deviation: 5 ppm; Maximum tolerated retention time shift: ± 0.10 minutes.

All statistical analyses were implemented in the R programming language for statistical computing ([91]; version 3.5.3). Only the most abundant features (i.e., ion) per metabolite were used for statistical analysis, which accounted to 364 N-containing metabolites. Of these, 26 metabolites were removed as these were not detected in at least three culture samples. Only the abundances of the native, monoisotopic features were utilized. Missing values were replaced by an abundance of 10,000. The imputed data matrix was auto-scaled. For univariate comparison, a global significance threshold of α = 0.05 and fold change (FC) ≥ 2.0 were used, while Sidak multiple testing correction (α = 0.00015) and FC ≥ 2.0 were used to classify metabolites as highly significantly different between the tested experimental conditions. For the heatmap illustration, the squared Euclidian distance and the Ward linkage method were applied.

Yeast complementation assay

T. atroviride npr1 was amplified from cDNA as template using primers TaNPR1_cDNA_fw and TaNPR1_cDNA_rev (S5 File). Primers YNpr1_Pro_fw and YNpr1_Pro_rev were used to amplify the promoter and primers TaNPR1_Ter_fw and TaNPR1_Ter_rev (S5 File) to amplify the terminator regions of the S. cerevisiae npr1 gene using genomic DNA of strain 23344c as template. Fragments were combined in vector YEplac195 [92] using the NEB® PCR Cloning Kit (New England Biolabs, Ipswich, UK) and the final plasmid (YEplac195-TaNpr1) was verified by sequencing. Yeast cells, the strains 23344c (ura3) and PVV357 (npr1Δ ura3) [8], were transformed with the indicated plasmids, as described previously [93]. For growth tests, cells were grown in minimal buffered (pH 6.1) medium containing 3% glucose as the carbon source and glutamate 0.1%, ammonium 1 mM, proline 0.1%, urea 0.1% or 1 mM, citrulline 0.05% or tryptophan 0.05% as the nitrogen sources [94].

Transcriptome analysis by RNA sequencing

T. atroviride was pre-cultivated for 48 hours in MM with 50 mM ammonium sulfate on a rotary shaker at 25°C starting from an inoculum of 2x106 spores/ml. Washed fungal biomass was then transferred into MM without nitrogen and starved for 5 hours before replacement to fresh MM with 50 mM ammonium sulfate. Three replicates each were treated with 20 μg/ml rapamycin or an equal volume of DMSO for 2 hours. Total RNA was isolated using RNeasy Plant mini kit (Qiagen, Venlo, Netherlands), following manufacturer’s instructions. Final elution was performed in Tris 10 mM, pH 7 (Thermo Fisher Scientific, Waltham, Massachusetts, USA). Illumina TruSeq mRNA library preparation, sequencing and differential gene expression analyses were performed by Microsynth (Balgach, Switzerland) according to their dedicated analysis pipeline. Obtained sequence reads were mapped to the T. atroviride genome (https://mycocosm.jgi.doe.gov/Triat2/Triat2.home.html) using software Bowtie 2 [95] and Differentially Expressed Genes (DEGs) were identified using DESeq2 [96]. Read counts were normalized, the variance calculated based on the three biological replicates per condition, and statistical testing applied for the identification of DEGs that are significantly up- or downregulated. Four sets of DEGs were identified: 1. comparison of T. atroviride wild type (WT) transcriptomes when grown in presence of DMSO (solvent control) versus growth in presence of rapamycin; 2. comparison of transcriptomes of the Δrhe2 mutant grown in presence of DMSO (solvent control) versus growth in presence of rapamycin; 3. comparison of transcriptomes of the WT grown in presence of DMSO versus Δrhe2 grown in presence of DMSO; 4. comparison of transcriptomes of the WT grown in presence of rapamycin versus Δrhe2 grown in presence of rapamycin (S6 Fig). Identified DEGs were considered as significant if p-adjusted value was ≤ 0.05 and were visualized as heatmap generated by using R package “ggplot2”. In order to identify shared and common DEGs among comparisons, Venn diagrams showing intersections of the four comparisons were generated through the R package “ggvenn”, and shared/common set of DEGs were listed. DEGs were associated by their IDs in the reference genome and the sequences were annotated with Blast2GO v.5 [97] to determine the related Gene Ontology (GO) terms. At the same time, all gene models of T. atroviride reference genome were annotated through Blast2GO to obtain an updated functional annotation. Fisher enrichment tests for significant DEGs obtained from the four comparisons were performed with Blast2GO to search for significant differences (False Discovery Rate ≤ 0.05) in frequencies of GO terms compared to all T. atroviride gene models (over representation). Results of Fisher enrichment test was slimmed in REVIGO [98]. The R package "GOplot” v. 1.0.2 was used to visualize the enriched GO terms in the four comparisons as bubble plots. For Fisher enrichment results, the z-score for each enriched category was calculated with the following formula: zscore = (up−down)/√count, where ‘up’ and ‘down’ are the number of assigned genes up-regulated (logFC > 0) or down- regulated (logFC < 0), respectively, and ‘count’ is the number of genes assigned to a term.

Identification of TOR pathway components

T. atroviride orthologues of proteins associated with the TOR signaling pathway were identified by BLASTp searches of the T. atroviride genome database (https://mycocosm.jgi.doe.gov/Triat2/Triat2.home.html) using default settings and the following queries: S. cerevisiae Tor1p (Saccharomyces Genome Database, SGD: YJR066W), S. cerevisiae Tor2p (SGD: YKL203C), S. cerevisiae Kog1p (SGD: YHR186C), S. cerevisiae Lst8p (YNL006W), S. cerevisiae Avo1p (SGD: YOL078W), S. cerevisiae Avo2p (SGD: YMR068W), S. cerevisiae Avo3p (SGD: YER093C), S. cerevisiae Rhb1p (SGD: YCR027C), S. cerevisiae Npr1p (SGD: YNL183C), F. fujikuroi Ff Npr1-1, FfNpr1-2 and FfNpr1-3 [43], S. pombe Tsc1 (PomBase: SPAC22F3.13) and Tsc2 (PomBase: SPAC630.13c).

Supporting information

S1 File.

(S1 Table) Mean ± standard deviation (cm) of colony diameter of T. atroviride wild type on PDA or minimal medium with 50 mM ammonium sulfate amended with rapamycin, torin1 or a rapamycin and torin1 combination as shown in Fig 1. Values which do not share at least one letter are statistically different within each column according to Tukey’s test (ANOVA). (S2 Table) Putative TOR pathway components encoded in the T. atroviride genome. Protein IDs were assigned by reciprocal best hit Blast using the given queries retrieved from the S. cerevisiae genome database (SGD; http://www.yeastgenome.org), the S. pombe genome database (PomBase; https://www.pombase.org), or [43] in case of F. fujikuroi. (S3 Table) Biomass (mg) of T. atroviride wild type and Δnpr1, Δrhe2, Δtsc1 and Δtsc2 mutants grown for 72 h in shake cultures in PBD or minimal medium amended with either 10 mM ammonium sulfate, L-glutamine or L-proline. Values which do not share at least one letter are statistically different within each row according to Tukey’s test (ANOVA).

https://doi.org/10.1371/journal.pone.0262180.s001

(XLSX)

S2 File. List of top 10 differentially expressed genes in the four comparisons analyzed.

https://doi.org/10.1371/journal.pone.0262180.s002

(XLSX)

S3 File. List of up-regulated genes in the different comparisons shown in the Venn diagram (Fig 8A).

https://doi.org/10.1371/journal.pone.0262180.s003

(XLS)

S4 File. List of down-regulated genes in the different comparisons shown in the Venn diagram (Fig 8B).

https://doi.org/10.1371/journal.pone.0262180.s004

(XLS)

S5 File. Primers used for generation of transformation cassettes, genotypic verification, and npr1 complementation.

https://doi.org/10.1371/journal.pone.0262180.s005

(XLSX)

S1 Fig.

(A) Southern blot analysis to confirm single copy integration of the hph deletion cassette in the Δtsc1- 11bIC mutant. Genomic DNA was digested with SacI or NheI followed by hybridization with a DIG-labelled probe of the hph gene. (B) qPCR analysis of hph copy number in different Δrhe2, Δnpr1, Δtsc1 and Δtsc2 mutants and the wild type control. Fold changes were normalized to the single copy in the Δtsc1-11bIC mutant. Mutant strains used for further experiments are marked with an asterisk.

https://doi.org/10.1371/journal.pone.0262180.s006

(TIF)

S2 Fig. Phenotype of different strains of Δnpr1, Δrhe2, Δtsc1 and Δtsc2 mutants and the T. atroviride wild type after 10 days of growth on PDA at 25°C.

https://doi.org/10.1371/journal.pone.0262180.s007

(TIF)

S3 Fig. Phenotype of different strains of Δnpr1, Δrhe2, Δtsc1 and Δtsc2 mutants and the T. atroviride wild type on different nitrogen sources.

Fungi were grown on minimal medium amended with selected nitrogen sources (10 ammonium sulfate, 10 mM L-glutamine, 10 mM sodium nitrate, 10 mM L-proline, or 10 mM urea) at 25°C for three days.

https://doi.org/10.1371/journal.pone.0262180.s008

(TIF)

S4 Fig. Joining cluster analysis from different time points (24, 48, 72 hours) of growth of T. atroviride wild type on Biolog PM3b nitrogen MicroPlates for evaluating the nitrogen source utilization capacity of the fungus.

https://doi.org/10.1371/journal.pone.0262180.s009

(TIF)

S5 Fig. Plate confrontation assays for assessing the mycoparasitic activity of T. atroviride Δnpr1, Δrhe2, Δtsc1 and Δtsc2 mutants and the wild type against R. solani.

Fungi were inoculated on opposite sides of an agar plate containing minimal medium with 50 mM ammonium sulfate (left side: T. atroviride; right side: R. solani) and grown at 25°C for 7 days. Pictures were taken after three and seven days.

https://doi.org/10.1371/journal.pone.0262180.s010

(TIF)

S6 Fig. 2D-plot (m/z versus retention time) of MetExtract-derived metabolic features.

Red: all metabolite ions detected in any of the four tested fungal strains. Blue: metabolite ions that carry at least one nitrogen atom in their molecular structure (nitrogen origination from NH4+ adducts was not considered).

https://doi.org/10.1371/journal.pone.0262180.s011

(TIF)

S7 Fig. Western blot analysis of ribosomal protein S6 (RPS6) phosphorylation in T. atroviride wild type and mutants Δnpr1, Δtsc1, Δtsc2, and Δrhe2 after treatment with 20 μg/ml rapamycin (+) or DMSO (-; mock control) for two hours.

Samples were probed for P-RPS6, total RPS6, and histone H3 as loading control using antibodies anti-phospho-(Ser/Thr) Akt substrate, anti-Rps6, and anti-H3.

https://doi.org/10.1371/journal.pone.0262180.s012

(TIF)

S8 Fig. Global transcriptomic response to TOR kinase inhibition.

(A) Scheme of the four computed comparisons for transcriptome analyses. (B) Heatmap of DEGs emerging from the four comparisons. Hierarchical clustering led to the identification of two main subclusters with comparisons 1 and 3 and comparisons 2 and 4 clustering together under the conditions tested.

https://doi.org/10.1371/journal.pone.0262180.s013

(TIF)

Acknowledgments

We thank Manuel Sanchez Lopez-Berges (University of Cordoba, Department of Genetics, Cordoba, Spain) and Daniel Flatschacher (University of Innsbruck, Department of Microbiology) for assistance with antibody selection and Western blotting, and Maraike Probst (University of Innsbruck, Department of Microbiology) for assistance with statistical analysis.

Citation: Segreto R, Bazafkan H, Millinger J, Schenk M, Atanasova L, Doppler M, et al. (2021) The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride. PLoS ONE 16(12): e0262180. https://doi.org/10.1371/journal.pone.0262180

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