Table 1.
Trichoderma species used for Eng18B sequencing, and translated amino acid positions used for Reverse Conservation Analysis.
Figure 1.
Trichoderma atroviride Eng18B homology model.
Ribbon diagrams of the catalytic module of Eng18B, based on the structure of T. reesei Eng18A (PDB entry 4AC1) showing the conserved catalytically important residues in red; variable regions from reverse conservation analysis (Wmeans) in green; and highly variable amino acid positions with Sscore ≥3 in blue. Variable regions are marked in Roman numerals from N- to C- termini.
Figure 2.
Expression analysis of the T. atroviride Eng18B gene.
Total RNA was extracted from T. atroviride mycelia after 24 h of incubation in submerged shake flask cultures at 25°C in darkness representing different nutritional/stress and mycoparasitic conditions. (A) T. atroviride Eng18B expression in glucose, C limitation, N limitation, C+N limitation, N-acetylglucosamine (GlcNAc), R. solani cell wall material (RsCW) and colloidal chitin mediums. (B) Eng18B expression 24 h after contact with R. solani (Ta-Rs24h) or B. cinerea (Ta-Bc24h). T. atroviride confronted with itself was used as control (Ta-Ta24h). Relative expression levels for Eng18B in relation to actin expression were calculated from the Ct values and the primer amplification efficiencies by using the formula described by Pfaffl [42]. Error bars represent standard deviation based on three biological replicates. Experiments in panel A and B were analysed separately, different letters indicate statistically significant differences (P≤0.05) within experiments.
Figure 3.
Colony morphology of WT, ΔEng18B and ΔEng18B+ T. atroviride strains in different nutrient regimes.
T. atroviride strains were inoculated on solid PDA, SMS, C limitation, N limitation, C+N limitation, N-acetylglucosamine (GlcNAc), chitin and glucose medium. Photographs of representative plates were taken 7 days post inoculation after incubation at 25°C. The experiments were carried out in three biological replicates.
Figure 4.
Conidiation of WT, ΔEng18B and ΔEng18B+ T. atroviride strains in different nutrient regimes.
T. atroviride strains were inoculated on solid PDA, SMS, C limitation, N limitation, C+N limitation, N-acetylglucosamine (GlcNAc), chitin and glucose medium and incubated at 25°C in darkness for 7 days, with daily light exposures to induce conidiation. Conidial numbers were determined using a Bright-Line haemocytometer as per instruction of manufacturer. Error bars represent standard deviation based on three biological replicates. Different letters indicate statistical significance (P≤0.05) for strain differences within a single medium.
Figure 5.
Growth rate of WT, ΔEng18B and ΔEng18B+ T. atroviride strains in different nutrient regimes.
T. atroviride strains were inoculated on solid PDA, SMS, C limitation, N limitation, C+N limitation, N-acetylglucosamine (GlcNAc), chitin and glucose medium and incubated at 25°C in darkness. Growth rate was calculated from data recorded 3 days post inoculation. Error bars represent standard deviation based on three biological replicates. Different letters indicate statistical significance (P≤0.05) for strain differences within a single medium.
Figure 6.
Growth rate of WT, ΔEng18B and ΔEng18B+ T. atroviride strains in abiotic stress medium.
T. atroviride strains were inoculated on solid PDA plates supplemented with 1 M NaCl or 0.025% (w/v) SDS and incubated at 25°C in darkness. Growth rate was calculated from data recorded 7 days post inoculation. Error bars represent standard deviation based on three biological replicates. Different letters indicate statistical significance (P≤0.05) for strain differences within a single medium.
Figure 7.
In vitro plate confrontation assays of WT, ΔEng18B and ΔEng18B+ T. atroviride strains.
(A) Plate confrontation against B. cinerea. Agar plugs of T. atroviride (right side in the plate) and B. cinerea (left side in the plate) were inoculated on opposite sides in 9 cm SMS agar plates and incubated at 25°C in darkness. The experiment was performed in three replicates and photographs of representative plates were taken 15 days post inoculation. (B and D) Secretion assay of B. cinerea. Agar plugs of B. cinerea was inoculated on SMS agar plates covered with cellophane and incubated at 25°C in darkness. After reaching the same diameter the colony was removed together with the cellophane disc and the plates re-inoculated with a T. atroviride WT, ΔEng18B or ΔEng18B+ agar plug and incubated at 25°C in darkness. Growth rate was calculated from data recorded 5 days post inoculation. The experiment was performed in three replicates and photographs of representative plates were taken 5 days post inoculation. (C and E) Secretion assay of WT, ΔEng18B and ΔEng18B+ T. atroviride strains. Agar plugs of T. atroviride WT, ΔEng18B or ΔEng18B+ strains were inoculated on SMS agar plates covered with cellophane and incubated at 25°C in darkness. After reaching the same diameter the colony was removed together with the cellophane disc and the plates re-inoculated with a B. cinerea agar plug and incubated at 25°C in darkness. Growth rate was calculated from data recorded 5 days post inoculation. The experiment was performed in three replicates and photographs of representative plates were taken 5 days post inoculation. Different letters indicate statistically significant differences (P≤0.05) within experiments.