Histone acetyltransferase TGF-1 regulates Trichoderma atroviride secondary metabolism and mycoparasitism

Abstract

Some filamentous fungi of the Trichoderma genus are used as biocontrol agents against airborne and soilborne phytopathogens. The proposed mechanism by which Trichoderma spp. antagonizes phytopathogens is through the release of lytic enzymes, antimicrobial compounds, mycoparasitism, and the induction of systemic disease-resistance in plants. Here we analyzed the role of TGF-1 (Trichoderma Gcn Five-1), a histone acetyltransferase of Trichoderma atroviride, in mycoparasitism and antibiosis against the phytopathogen Rhizoctonia solani. Trichostatin A (TSA), a histone deacetylase inhibitor that promotes histone acetylation, slightly affected T. atroviride and R. solani growth, but not the growth of the mycoparasite over R. solani. Application of TSA to the liquid medium induced synthesis of antimicrobial compounds. Expression analysis of the mycoparasitism-related genes ech-42 and prb-1, which encode an endochitinase and a proteinase, as well as the secondary metabolism-related genes pbs-1 and tps-1, which encode a peptaibol synthetase and a terpene synthase, respectively, showed that they were regulated by TSA. A T. atroviride strain harboring a deletion of tgf-1 gene showed slow growth, thinner and less branched hyphae than the wild-type strain, whereas its ability to coil around the R. solani hyphae was not affected. Δtgf-1 presented a diminished capacity to grow over R. solani, but the ability of its mycelium -free culture filtrates (MFCF) to inhibit the phytopathogen growth was enhanced. Intriguingly, addition of TSA to the culture medium reverted the enhanced inhibition growth of Δtgf-1 MFCF on R. solani at levels compared to the wild-type MFCF grown in medium amended with TSA. The presence of R. solani mycelium in the culture medium induced similar proteinase activity in a Δtgf-1 compared to the wild-type, whereas the chitinolytic activity was higher in a Δtgf-1 mutant in the absence of R. solani, compared to the parental strain. Expression of mycoparasitism- and secondary metabolism-related genes in Δtgf-1 was differentially regulated in the presence or absence of R. solani. These results indicate that histone acetylation may play important roles in the biocontrol mechanisms of T. atroviride.

Fig 1. Trichostatin A did not affect the overgrowth of Trichoderma atroviride on Rhizoctonia solani.

Dual confrontation plates of T. atroviride (left side) against R. solani (right side) on PDA plates in the absence (A) or presence (B) of 300 nM Trichostatin A, incubated at 28 °C and photographed at 36, 48, 60, 72, and 96 h. Ta = T. atroviride, Rs = R. solani, Trichostatin A = TSA. Images are representative of similar results from three independent trials.

Fig 2. TSA enhances the antibiotic activity of T. atroviride mycelium-free culture filtrates (MFCF) against R. solani.

T. atroviride was grown in PDB amended or not with 300 nM TSA, incubated for 7 days at 28 °C. The MFCFs were added to PDA 1× medium at a final concentration of 60%. R. solani radial growth was measured at 12, 24, 36, 48, and 60 h post-inoculation. R. solani growth on PDA (black bars), PDA plates amended with 300 nM TSA (arrow filled bars), PDA plates amended with 60% of T. atroviride MFCF (crosshatched bars), and PDA plates amended with 60% of T. atroviride MFCF grown in PDB plus TSA (black dotted bars) are shown. The bars show the mean ± SD of three independent biological replicates. Different letters are used to indicate means that differ significantly (P < 0.05). Three replicate plates were established for each treatment, and the experiment was repeated twice.

Fig 3. TSA and R. solani presence negatively affected T. atroviride ech-42, prb-1, pbs-1, and tps-1 gene expression.

T. atroviride and R. solani were co-cultured on PDA (A, B, C, and D) or PDA amended with 300 nM TSA (E, F, G, and H) at 28 °C. Total RNA was extracted from T. atroviride mycelium collected at 36, 48, and 60 h. Relative expression was calibrated using act-1 as housekeeping gene and normalized against the wt strain in the absence of the phytopathogen. ech-42 = 42-kDa endochitinase gene, prb-1 = basic proteinase gene, pbs-1 = peptaibol synthase gene, and tps-1 = terpene synthase gene. Black bars, T. atroviride wt strain growing alone (Ta); grey bars, T. atroviride vs R. solani (Ta+Rs) co-cultures. The bars show the mean ± SD of three independent biological replicates. Different letters are used to indicate means that differ significantly (P < 0.05). This assay was repeated twice per triplicate with similar results.

Fig 4. Colony morphology and development of T. atroviride wt and Δtgf-1 strains.

T. atroviride wt (A) and Δtgf-1 (B) were grown on PDA plates for 7 days at 28 °C and photographed. Both strains were co-cultured on PDA medium, fixed, dehydrated, and analyzed by SEM at 100 (C) and 400 (D) magnifications. The wt is shown in the right side whereas the Δtgf-1 is shown in the left side of the photomicrographs. Images are representative of similar results from two independent trials, including three replicate plates.

Fig 5. Dual culture assays of T. atroviride wt and Δtgf-1 strains versus R. solani.

wt and Δtgf-1 strains were inoculated at the left side of the Petri dish, whereas R. solani was inoculated at the right side. Dual cultures were incubated at 28 °C, and photographed at 72, 96, 120, 168, and 192 h. Ta = T. atroviride wt strain, Δtgf-1 = T. atroviride tgf-1 mutant strain, Rs = Rhizoctonia solani. This assay was repeated twice including three replicate plates.

Fig 6. Deletion of tgf-1 did not affect T. atroviride mycoparasitic effect against R. solani.

Dual culture assays of T. atroviride wt (A) and Δtgf-1 (B) strains versus R. solani were analyzed by SEM (C and D, respectively). Blue arrowheads show R. solani hyphae, whereas red arrowheads show wt and Δtgf-1 coiling hyphae on R. solani. SEM photomicrographs were magnified 1500 ×. The experiments were repeated twice and representative photographs are shown.

Fig 7. Antagonistic ability of Δtgf-1 strain against other Trichoderma species.

Dual cultures of T. atroviride wt and Δtgf-1 strains against other species of T. atroviride, indicated on the right side of the figure, were grown at 28 °C for 96 h. Ta = T. atroviride, Δtgf-1 = T. atroviride tgf-1 mutant strain, Tv = T. virens, Tc = T. citrinoviride, Th = T. harzianum. Images are representative of similar results from three independent trials.

Fig 8. Antibiosis assay using MFCF obtained from T. atroviride wt or Δtgf-1 strains grown in the presence or absence of TSA against R. solani.

The Δtgf-1 and wt strains were grown for 7 days in PDB medium amended or not with 300 nM TSA, at 28 °C. MFCFs obtained from each of these cultures were added to PDA 1× medium at a final concentration of 60%. R. solani was inoculated into the different media and its radial growth was determined at 12, 24, 36, 48, and 60 h. Radial growth of R. solani on PDA containing T. atroviride wt strain MFCF without TSA (black bars) or amended with TSA (crosshatched bars) was determined at the indicated times. Radial growth of R. solani on PDA containing T. atroviride Δtgf-1 strain MFCF without TSA (black dotted bars) or with TSA (arrow filled bars) was determined at the indicated times. The bars show the mean ± SD of three independent biological replicates. Different letters are used to indicate means that differ significantly (P < 0.05). Three replicate plates were established for each treatment, and the experiment was repeated twice.

Fig 9. TGF-1 plays a negative role in chitinolytic activity but a minor role in proteolytic activity in T. atroviride.

Chitinolytic activity was determined using colloidal chitin by estimating the reducing sugars with dinitro-salicylic acid (DNS) method, measuring absorbance spectrophotometrically at 540 nm. One unit of chitinase activity was defined as the amount of enzyme required to increase absorbance at 540 nm by 1 OD unit ml^-1 h^-1. Proteinase activity was determined using mM Suc-Ala-Ala-Pro-Phe-pNA (0.5 mM) as substrate, the liberation of pNA was measured spectrophotometrically at 405 nm. The activity was expressed as p-nitroanilide released per min. Specific activity was referred to 1 mg of protein. The effect of induced MFCF of the wt and Δtgf-1 strains was tested over both activities. The bars show the mean ± SD of three independent biological replicates. Different letters are used to indicate means that differ significantly (P < 0.05). Chitinolytic and proteolytic activities were tested in three replicates and repeated two times.

Fig 10. TGF-1 and TSA differentially regulate T. atroviride ech-42, prb-1, pbs-1 and tps-1 in the presence or absence of R. solani.

Dual culture assays of T. atroviride wt and Δtgf-1 against R. solani, or grown alone as control were performed on PDA in absence (A, B, C, D) or in presence of 300 nM TSA (E, F, G, H) at 28 °C. Total RNA was extracted from T. atroviride mycelium collected at 36, 48, and 60 h. Relative expression was calibrated using act-1 as housekeeping gene and normalized against the wt strain in absence of the phytopathogen. ech-42 = 42-kDa endochitinase gen, prb-1 = basic proteinase gene, pbs-1 = peptaibol synthase gene, tps-1 = terpene synthase gene. Black bars represent Δtgf-1 strain, whereas gray bars represent Δtgf-1 vs R. solani interaction. Addition of TSA is indicated at the top of the panels. The bars show the mean ± SD of three independent biological replicates. Different letters are used to indicate means that differ significantly (P < 0.05). This assay was repeated twice per triplicate with similar results.