Fig 1.
Phylogenetic and structural classification of chitin synthases (chs) and -deacetylases (cda) in Trichoderma atroviride.
Structure/function prediction of the (A) eight chitin synthases (CHS1-8) and (B) six chitin deacetylases (CDA1-6). On the left side a condensed phylogenetic tree (dendrogram) is shown for both enzyme groups. (C) Phylogeny and functional relation of all six identified chitin deacetylases from T. atroviride in comparison to other organisms. Three functional groups are highlighted: light blue, virulence/autolysis related chitin deacetylases; light green/green, vegetative and conidial development-related transmembrane CDAs group I/ II; yellow, CDAs putatively involved in self-/nonself recognition. Bootstraps above 80 are not indicated. The bar marker indicates genetic distance. Gene name abbreviations are described in S1 Fig. A complete phylogeny of CHS1-8 in filamentous fungi is depicted in the S1 Fig.
Fig 2.
Genomic environment of chs and cda reflects their specific roles.
(A, B) A cluster analysis was performed according to the chromosomal location of chitin synthase genes and their conservation among T. atroviride (Ta) and T. reesei (Tr) annotated at the JGI database for Trichoderma atroviride v2.0 (https://genome.jgi.doe.gov/Triat2/Triat2.home.html) and Trichoderma reesei v2.0 (https://genome.jgi.doe.gov/Trire2/Trire2.home.html). Conservation of exons (lilac), UTRs (untranslated regions, turquoise) and CNS (conserved non-coding sequences, red) is shown in the above panel of each diagram. Gene location on the chromosomes (T. reesei, [146]) and contigs (T. atroviride) is indicated in k = 1,000 nucleotides. Gene (white arrows) size and position are to scale and numbers correspond to gene description in S1 and S2 Tables. Chitin synthase genes and the activator are highlighted in red. (A) Gene cluster of chitin synthase (chs4) and the putative chitin synthase activator (csa1). (B) chs5-chs7-chs6 gene cluster. (C-D) Transcript levels of chitin synthase (left panel) and chitin deacetylase genes (right panel) during vegetative growth and conidiation. Transcript levels of chitin synthase genes (chs1-8) were related to chs1 and chitin deacetylase genes (cda1-6) to cda2 expression at 16 h of germination for (C) and 24 h of growth on PDA for (D). Normalized expression of chs1 and cda2 corresponds to ‘1’ as indicated (see also S2 Fig). (C) Expression analysis during germination after 0 h, 4 h (isotropic growth), 8 h (formation of germination tube) and 16 h (first vegetative hyphae). Pictures of corresponding germination stages are shown on the right-hand side; scale bar = 20μm. (D) Expression analysis during hyphal development from mycelium grown on PDA for 24 h and 48 h and asexual development at different maturation stages with (w) white/ nascent, (y) yellow/immature and (g) green/mature conidia; scale bar = 1 cm.
Fig 3.
Differential expression of chs and cda upon environmental stresses.
(A) Transcription analysis of chitin synthase (left panel) and chitin deacetylase (right panel) genes using various stressors. Comparison of growth under control conditions (glc1%, 1% glucose, ctrl) and with different stressors. On the left side the specific growth condition is indicated and on the right side a stress group is assigned: CWI (cell wall integrity disturbing stressors); CWSD (cell wall synthesis disturbing agents); HOG (high osmolarity glycerol) and ROS (reactive oxygen species). Detailed growth conditions are described in S3 Fig. The clustering results of transcription levels (values calculated by ΔΔCT method) are represented in ‘jet’-style color mapping. (B) Graphical representation of the 1,200 bp upstream promoter regions of the eight chitin synthases and the identified stress element binding regions. Color code boxes indicate various stress groups whose height corresponds to the score level. CWI, HOG, ROS see (A); DEV, developmental regulation; OX, oxygen stress related; STRESS, general stress element; TATA, TATA-box or CAAT-box. Other detected and enriched motifs are marked with an asterisk. For more details on the promoter response elements see S3 Table.
Fig 4.
Critical roles of chs and cda in growth and development are emphasized in single knockout mutants.
(A) Growth of generated deletion mutants and the T. atroviride wild type strain (WT) on PDA after 72 h. (B) Colony extension in percent of the parental strain (wt). Growth on PDA only and on plates containing 1.2 M sorbitol (+sorbit), 200 μg/ml calcofluor white (+CFW) or 150 μg/ml congo red (+CR) was compared. (C, D) Generation of asexual conidia by all deletion mutants, see also S4A Fig; (C) spores/cm2 and (D) average conidial size. (E-F) Compensatory transcription analysis of the transglycosylase genes crh1a, crh1b and crh2 in the Δchs (E) and Δcda (F) strains compared to expression in the WT strain after 25 h and 48 h of growth on PDA (tef1 used as housekeeping gene). Mean +/- SEM are shown and p>0.005 are indicated with *, upregulated; °, downregulated. (B-F) Data was generated from at least two independent experiments.
Fig 5.
Single knockout mutants show severe morphological changes and aberrant chitin deposition in patches with reduced chitin levels.
(A) Chitin deposition and tip morphology in leading hyphae of the peripheral zone of each colony was visualized by CFW staining in epifluorescence microcopy using the inverted agar method for live-cell imaging [91]; scale bar = 20μm. (B) CFW staining highlights aberrant chitin deposition and septal development in selected deletion mutants; scale bar = 10μm. (C) Altered cell wall chitin deposition in chs and cda deletion strains compared to the WT was investigated by semiquantitative fluorescence microscopy using CFW and CR as stains. A defined area of the tip apex and subapex (shown in B) of leading hyphae was analyzed by densitometry using the ImageJ software platform (http://rsb.info.nih.gov/ij/). Mean +/- SEM from n = 25–60 hyphae are shown.
Table 1.
Relative GlcNAc content in the cell wall of mutant strains compared to the wild type.
Fig 6.
Mycoparasitism depends on coordinated expression of chitin synthases,–deacetylases and chitosanases.
Differential transcription analysis of chitin synthases, chitin deacetylases and chitosanases during the mycoparasitic attack of T. atroviride against three different hosts. Expression of all target genes was analyzed using the ΔΔCT method. T. atroviride against itself (Ta), R. solani (Rs), S. sclerotiorum (Ssc) or B. cinerea (Bc). The WT grown alone on a plate (control condition) was arbitrarily set to ‘1’. Samples were collected at contact and after contact (4 mm overgrowth). The gene sar1 was used as the housekeeping gene. Data were generated from two independent experiments and three technical replicates. Mean +/- SEM and p>0.005 are indicated with *, upregulated; °, downregulated. (A) Transcription analysis in confrontation assays with fungal hosts, chs1-8, chitin synthase; cda1-6, chitin deacetylase. The clustering results are represented in ‘jet’-style color mapping as described in Fig 3A. (B) Relative expression of all identified chitosanase genes (cho1-cho6) during mycoparasitism.
Fig 7.
Cell wall chitin and chitosan affect the mycoparasitic attack and resistance to hosts.
(A) Dual confrontation assays: A 5 x 30 mm slice of fully overgrown PDA plates of the indicated T. atroviride WT and mutant strains (T) was placed on the left side of a PDA plate 5 cm apart from a slice of the host S. sclerotiorum (H, right side) and incubated for 5 days. Arrows indicate the zonal overgrowth (dotted line) by T. atroviride over S. sclerotiorum. (B) Microscopic analysis of the confrontation zone of. T. atroviride WT and selected mutant strains against S. sclerotiorum using CFW staining in the inverted agar method for live-cell imaging [91]; arrows indicate hyphal attachment; L, hyphal leakage; r, hyphal retreat; *, increased hyphal branching; scale bar indicated. (C) Plates from dual confrontation assay in (A) were photographed from the bottom to show the brownish contact zone, in cda mutants compared to the wild type. (D) Relative expression of mycoparasitic indicator genes, prb1, nag1, ech42 in the WT (wt, striped columns) and cda deletion (12356) mutants against S. sclerotiorum. Samples were collected at contact and after contact (4 mm overgrowth). WT alone (control condition) was arbitrarily set to ‘1’, (wt, striped columns). Expression data was normalized to the housekeeping gene sar1. Data were generated from two independent experiments and three technical replicates. Mean +/- SEM and p>0.005 are indicated with *, upregulated; °, downregulated. (E) Effects of antifungal metabolites and secreted enzymes from the host S. sclerotiorum on growth of T. atroviride WT and mutant strains. S. sclerotiorum was cultured on top of a diffusible cellophane membrane covering PDA plates and after 48 h of growth the cellophane disc was removed and plates containing the secreted active compounds were inoculated with an agar plug of Trichoderma WT and mutant strains. The percent growth inhibition by S. sclerotiorum secreted active extract was calculated from comparison with the growth of the respective strains on fresh PDA. A representative of three independent experiments is shown. (F) The susceptibility to oxidative stress of Δchs and Δcda strains was demonstrated on a solid medium containing H2O2. Colony extension in percent of the parental strain on minimal medium with (right panel) or without (left panel) the addition of 0.0075% H2O2 and growth for 72 h was determined. WT (white), chitin synthase mutants (grey), chitin deacetylase mutants (light grey) were compared. Mean +/- SEM from two independent experiments is shown.
Fig 8.
Specified roles of chitin-modifying enzymes during the life cycle of T. atroviride.
Schematic representation of the involvement of chitin synthases (chs, yellow), chitin deacetylases (cda, pink), chitosanases (cho, orange) and transglycosylases (crh, turquoise) in various stages of development of Trichoderma atroviride. Vegetative development, including germination, conidiophore formation, conidial maturation and self recognition, environmental stress response and mycoparasitism with invasion and feeding on a host cell wall, are depicted. The most important players are depicted in bold letters. The role of putatively secreted and membrane-bound enzymes in mycoparasitism (in association with secondary metabolites and other enzymes, not shown), exogenous chitin and chitosan degradation (released by the fungi), and in the remodeling and recycling of the own cell wall (self-recognition) and the host is demonstrated.