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.
Highlights
Several Trichoderma species are potent mycoparasites known as biocontrol agents for the protection of plants against fungal phytopathogens
On minimal media (MM) with ammonium sulfate as sole nitrogen source, rapamycin more strongly interfered with fungal growth than on potato dextrose agar (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
When cultivating T. atroviride on PDA supplemented with the second generation target of rapamycin (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
Summary
Several Trichoderma species are potent mycoparasites known as biocontrol agents for the protection of plants against fungal phytopathogens. The rapamycin-triggered TOR inhibition notably causes nuclear accumulation of the GATAtype transcription factor Gln 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 post-translationally regulated by the protein kinase Npr (nitrogen permease reactivator 1) [4,5,6,7]. In case of TOR inhibition by rapamycin, Npr becomes functional allowing the TOR pathway to rapidly adjust the permeability of nutrients to regulate cell growth in response to environmental nutrient availability [8,9,10]
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