Trichoderma atroviride transcriptional regulator Xyr1 supports the induction of systemic resistance in plants.

As a result of a transcriptome-wide analysis of the ascomycete Trichoderma atroviride, mycoparasitism-related genes were identified; of these, 13 genes were further investigated for differential expression. In silico analysis of the upstream regulatory regions of these genes pointed to xylanase regulator 1 (Xyr1) as a putatively involved regulatory protein. Transcript analysis of the xyr1 gene of T. atroviride in confrontation with other fungi allowed us to determine that xyr1 levels increased during mycoparasitism. To gain knowledge about the precise role of Xyr1 in the mycoparasitic process, the corresponding gene was deleted from the T. atroviride genome. This resulted in strong reductions in the transcript levels of axe1 and swo1, which encode accessory cell wall-degrading enzymes considered relevant for mycoparasitism. We also analyzed the role of Xyr1 in the Trichoderma-Arabidopsis interaction, finding that the plant response elicited by T. atroviride is delayed if Xyr1 is missing in the fungus.

Elevated activity of dolichyl phosphate mannose synthase enhances biocontrol abilities of Trichoderma atroviride.

Antagonism of Trichoderma spp. against phytopathogenic fungi is widely exploited for biocontrol of plant diseases. A crucial role in the biocontrol mechanism is attributed to cell-wall-degrading enzymes secreted by Trichoderma spp. Therefore, more efficient production and secretion of the enzymes should elevate the biocontrol abilities of Trichoderma spp. Because the majority of secretory hydrolases are glycoproteins, it has been postulated that the posttranslational modification of these proteins could constitute a bottleneck in their production and secretion. Our previous study showed that improvement of O-glycosylation elevated protein secretion by Trichoderma reesei. In this study, we enhanced the biocontrol abilities of T. atroviride P1 against plant pathogens by overexpressing the Saccharomyces cerevisiae DPM1 gene coding for dolichyl phosphate mannose (DPM) synthase, a key enzyme in the O-glycosylation pathway. The transformants we obtained showed doubled DPM synthase activity and, at the same time, significantly elevated cellulolytic activity. They also revealed an improved antifungal activity against the plant pathogen Pythium ultimum.

Identification of mycoparasitism-related genes in Trichoderma atroviride.

A high-throughput sequencing approach was utilized to carry out a comparative transcriptome analysis of Trichoderma atroviride IMI206040 during mycoparasitic interactions with the plant-pathogenic fungus Rhizoctonia solani. In this study, transcript fragments of 7,797 Trichoderma genes were sequenced, 175 of which were host responsive. According to the functional annotation of these genes by KOG (eukaryotic orthologous groups), the most abundant group during direct contact was "metabolism." Quantitative reverse transcription (RT)-PCR confirmed the differential transcription of 13 genes (including swo1, encoding an expansin-like protein; axe1, coding for an acetyl xylan esterase; and homologs of genes encoding the aspartyl protease papA and a trypsin-like protease, pra1) in the presence of R. solani. An additional relative gene expression analysis of these genes, conducted at different stages of mycoparasitism against Botrytis cinerea and Phytophthora capsici, revealed a synergistic transcription of various genes involved in cell wall degradation. The similarities in expression patterns and the occurrence of regulatory binding sites in the corresponding promoter regions suggest a possible analog regulation of these genes during the mycoparasitism of T. atroviride. Furthermore, a chitin- and distance-dependent induction of pra1 was demonstrated.

Profiling of trichorzianines in culture samples of Trichoderma atroviride by liquid chromatography/tandem mass spectrometry.

Peptaibols are bioactive linear peptides of 5-20 amino acid residues and contain specific non-proteinogenic amino acids such as alpha-aminoisobutyric acid (Aib). They are antibiotic secondary metabolites of moulds belonging predominantly to the genus Trichoderma, some species of which are successfully used as biocontrol organisms to fight against plant diseases. In the present study we developed a profiling method for the relative quantification of 16 trichorzianine peptaibols in culture samples of T. atroviride and the comparison of their expression patterns by liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS). The method is based on selected reaction monitoring (SRM) in a triple-quadrupole tandem mass spectrometer using three SRM transitions per compound. The trichorzianines were enriched by solid-phase extraction (SPE) on C(18) cartridges. SPE recoveries were evaluated for diluted trichorzianine standard solutions and ranged from 72-97%. Suppression of the ionisation of the peptaibols in the ESI source ranged from 67-128% for most of the trichorzianines in culture filtrates of two different strains of T. atroviride and in spiked culture medium. In the case of trichorzianines TA Vb, TA VIa and TA VIb the presence of matrix components in the fungal culture samples caused a reduction of the SRM signal, with intensities between 34% and 56% relative to pure standard solutions. Finally, the profiling method was successfully applied to culture samples of T. atroviride P1 wild-type and two deletion mutants showing different trichorzianine expression patterns characteristic for the investigated fungal strains. This is the first LC-SRM profiling method for peptaibols for the investigation of peptaibol expression patterns in fungal culture samples.

Signaling via the Trichoderma atroviride mitogen-activated protein kinase Tmk 1 differentially affects mycoparasitism and plant protection.

Trichoderma atroviride is a mycoparasite of a number of plant pathogenic fungi thereby employing morphological changes and secretion of cell wall degrading enzymes and antibiotics. The function of the tmk 1 gene encoding a mitogen-activated protein kinase (MAPK) during fungal growth, mycoparasitic interaction, and biocontrol was examined in T. atroviride. Deltatmk 1 mutants exhibited altered radial growth and conidiation, and displayed de-regulated infection structure formation in the absence of a host-derived signal. In confrontation assays, tmk 1 deletion caused reduced mycoparasitic activity although attachment to Rhizoctonia solani and Botrytis cinerea hyphae was comparable to the parental strain. Under chitinase-inducing conditions, nag 1 and ech 42 transcript levels and extracellular chitinase activities were elevated in a Deltatmk 1 mutant, whereas upon direct confrontation with R. solani or B. cinerea a host-specific regulation of ech 42 transcription was found and nag 1 gene transcription was no more inducible over an elevated basal level. Deltatmk 1 mutants exhibited higher antifungal activity caused by low molecular weight substances, which was reflected by an over-production of 6-pentyl-alpha-pyrone and peptaibol antibiotics. In biocontrol assays, a Deltatmk 1 mutant displayed a higher ability to protect bean plants against R. solani.

The G protein alpha subunit Tga1 of Trichoderma atroviride is involved in chitinase formation and differential production of antifungal metabolites.

Trichoderma mycoparasitism includes recognition, attack, overgrowth and lysis of the host fungus accompanied by morphological changes and secretion of hydrolytic enzymes and antibiotics. Studying the underlying signal transduction pathways, the tga1 gene encoding a Galpha subunit of Trichoderma atroviride P1 was analysed. A Deltatga1 mutant showed continuous sporulation and elevated internal steady-state cAMP levels. tga1 gene deletion resulted in a complete loss of mycoparasitic overgrowth and lysis of Rhizoctonia solani, Botrytis cinerea, and Sclerotinia sclerotiorum during direct confrontation, although infection structure formation was unaffected. The reduced mycoparasitic abilities were reflected by strongly decreased chitinase activities and reduced nag1 and ech42 gene transcription. Furthermore, production of 6-pentyl-alpha-pyrone and of metabolites with sesquiterpene structure was reduced in the Deltatga1 mutant. Regardless of these deficiencies, the mutant displayed an enhanced growth inhibition of the host fungi by over-producing other low molecular weight antifungal metabolites, suggesting opposite roles of Tga1 in regulating the biosynthesis of different antifungal substances in T. atroviride.

Signal transduction by Tga3, a novel G protein alpha subunit of Trichoderma atroviride.

Trichoderma species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when Trichoderma specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the tga3 gene of Trichoderma atroviride, which encodes a novel G protein alpha subunit that belongs to subgroup III of fungal Galpha proteins. Deltatga3 mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with Rhizoctonia solani or Botrytis cinerea, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or N-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes ech42 and nag1 were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus, T. atroviride Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.