Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis

Abstract

Trichoderma species belong to a class of free-living fungi beneficial to plants that are common in the rhizosphere. We investigated the role of auxin in regulating the growth and development of Arabidopsis (Arabidopsis thaliana) seedlings in response to inoculation with Trichoderma virens and Trichoderma atroviride by developing a plant-fungus interaction system. Wild-type Arabidopsis seedlings inoculated with either T. virens or T. atroviride showed characteristic auxin-related phenotypes, including increased biomass production and stimulated lateral root development. Mutations in genes involved in auxin transport or signaling, AUX1, BIG, EIR1, and AXR1, were found to reduce the growth-promoting and root developmental effects of T. virens inoculation. When grown under axenic conditions, T. virens produced the auxin-related compounds indole-3-acetic acid, indole-3-acetaldehyde, and indole-3-ethanol. A comparative analysis of all three indolic compounds provided detailed information about the structure-activity relationship based on their efficacy at modulating root system architecture, activation of auxin-regulated gene expression, and rescue of the root hair-defective phenotype of the rhd6 auxin response Arabidopsis mutant. Our results highlight the important role of auxin signaling for plant growth promotion by T. virens.

Figure 1.

Effects of T. virens and T. atroviride inoculation on the growth of Arabidopsis seedlings. A, Photograph of 9-d-old Arabidopsis (Col-0) seedlings grown on the surface of agar plates containing 0.2× MS medium. Seedlings were treated with sterilized water at day 4 and photographed 5 d later. Bar = 1 cm. B, Representative photograph of Arabidopsis seedlings that were inoculated with T. virens at a distance of 5 cm from the root tip at 4 d after germination and grown for a further 5-d period. C, Photograph of Arabidopsis seedlings inoculated with T. atroviride at a distance of 5 cm from the root tip at 4 d after germination and grown for a further 5-d period. D, Effects of fungal inoculation on shoot biomass production. Photographs show representative individuals of four plates per treatment. Data from D show means ± sd from three groups of 10 seedlings that were recovered from the medium, excised at the root/shoot junction, and weighed on an analytical scale. Different letters represent means statistically different at the 0.05 level. The experiment was repeated three times with similar results. [See online article for color version of this figure.]

Figure 2.

Effects of Trichoderma inoculation on Arabidopsis root system architecture. Arabidopsis Col-0 seedlings were germinated and grown for 4 d on the surface of agar plates containing 0.2× MS medium. Half of the plates were inoculated with T. virens or T. atroviride at a distance of 5 cm from the primary root tip and grown for an additional 5-d period. A, Primary root length. B, Lateral root number per plant. C, Stage number of lateral root primordia per plant. D, Total lateral root primordia per plant. Values shown are means ± sd (n = 30). Different letters represent means statistically different at the 0.05 level. The experiment was repeated three times with similar results.

Figure 3.

Effects of T. virens inoculation on auxin-regulated gene expression. Twelve-hour GUS staining of DR5:uidA primary roots of Arabidopsis seedlings grown for 4 d on agar-solidified 0.2× MS medium. A to C, Uninoculated seedlings. D to F, T. virens-inoculated seedlings. Photographs show representative individuals of at least 20 stained seedlings. The experiment was repeated twice with similar results. Bars = 100 μm. [See online article for color version of this figure.]

Figure 4.

Effects of T. virens inoculation on biomass production and lateral root development in wild-type Arabidopsis (Col-0) and auxin-related mutants. Plant material was harvested 5 d after fungal inoculation. Shoots were excised at the root/shoot junction, and the fresh weight was determined on an analytical balance. A, Shoot fresh weight. B, Lateral root number per plant. Values shown represent means of four groups of 10 seedlings ± sd. Lateral roots were quantified for 30 seedlings. Different letters are used to indicate means that differ significantly (P < 0.05). The experiment was repeated three times with similar results.

Figure 5.

Determination of IAA from derivatized samples from T. virens growth medium by GC-MS. A, Total ion chromatogram of IAA from acidic ethyl acetate extract obtained from 1 L of culture medium of T. virens. B and C, The 70-eV electron-impact full-scan mass spectra from m/z 50 to 500 of IAA methyl ester identified in the extract (B) and the methylated IAA standard (C). Determinations were done from at least five independent samples.

Figure 6.

Determination of indolic compounds from underivatized samples from T. virens growth medium by GC-MS. A, Total ion chromatogram of IAAld and IEt from neutral ethyl acetate extract obtained from 1 L of culture medium of T. virens. B to E, The 70-eV electron-impact full-scan mass spectra from m/z 50 to 500 of IAAld identified in the extract (B), the standard IAAld (C), IEt identified in the extract (D), and the standard IEt (E). Determinations were done from at least five independent samples.

Figure 7.

Effects of indolic compounds produced by T. virens on auxin-regulated gene expression. A to H, Twelve-hour GUS staining of DR5:uidA Arabidopsis seedlings grown for 6 d on agar plates containing 0.2× MS medium (A and E) and on medium supplied with 2 μm IAA (B and F), 4 μm IAAld (C and G), or 64 μm IEt (D and H). Notice the increase in GUS expression in shoots and roots in the treatments with IAAld. I to P, Twelve-hour GUS staining of BA3:uidA Arabidopsis seedlings grown for 6 d on agar plates containing 0.2× MS medium (I and M) and on medium supplied with 2 μm IAA (J and N), 4 μm IAAld (K and O), or 64 μm IEt (L and P). Notice the increase in GUS expression in the root elongation region in the treatments with IAA or IAAld. Photographs are representative individuals of at least 20 stained seedlings. The experiment was repeated twice with similar results. DMSO, Dimethyl sulfoxide. [See online article for color version of this figure.]

Figure 8.

Analysis of Aux/IAA stability with HS∷AXR3NT-GUS fusions. Wild-type seedlings expressing the HS∷AXR3NT-GUS constructs were heat shocked at 37°C for 2 h. After heat induction, the seedlings were treated with IAA, IAAld, or IEt for different time periods at the indicated concentrations and stained overnight for GUS activity. Notice the degradation of the fusion protein by either IAA or IAAld. A to P, Representative photographs of cotyledons (n = 10 stained seedlings). Similar results were obtained in two independent experiments. [See online article for color version of this figure.]

Figure 9.

Effects of IAAld on Arabidopsis root architecture. Wild-type Col-0 seedlings were grown for 10 d under increasing IAAld concentrations on vertically oriented agar plates. Data are given for the length of the primary root (A), lateral root number (B), and lateral root density (C). Values shown represent means of 30 seedlings ± sd. Different letters represent means statistically different at the 0.05 level. The experiment was repeated three times with similar results.

Figure 10.

Effects of IEt on Arabidopsis root architecture. Wild-type Col-0 seedlings were grown for 12 d under increasing IEt concentrations on vertically oriented agar plates. Data are given for the length of the primary root (A), lateral root number (B), and lateral root density (C). Values shown represent means of 30 seedlings ± sd. Different letters represent means statistically different at the 0.05 level. The experiment was repeated three times with similar results.

Figure 11.

IAAld rescues the rhd6 mutant phenotype. A, Wild-type Col-0 Arabidopsis root with normal root hair formation. B and C, Root hair formation in response to IAA (B) or IAAld (C) treatment. D, A typical rhd6 Arabidopsis mutant root showing a reduction in root hair formation. E and F, Formation of root hairs in rhd6 roots in response to IAA (E) or IAAld (F) treatment. The experiment was repeated three times with similar results. DMSO, Dimethyl sulfoxide. [See online article for color version of this figure.]

Figure 12.

Effects of IAA on Arabidopsis biomass production. Wild-type Col-0 seedlings were grown for 14 d under increasing IAA concentrations on vertically oriented agar plates. Data are given for the mean root fresh weight (A), shoot fresh weight (B), and total fresh weight (C). Plants were excised at the root/shoot junction, and fresh weights were determined on an analytical scale for four groups of 25 plants. Different letters represent means statistically different at the 0.05 level. The experiment was repeated twice with similar results.

Figure 13.

Arabidopsis growth responses to T. virens and their regulation. T. virens induces lateral root proliferation and enhances biomass accumulation by production of IAA and IAAld. IAAld can be converted to IAA by plant enzymes or can directly regulate auxin-inducible gene expression, possibly by interacting with auxin receptors. IEt did not show clear auxin-like activity, but it can act as a storage form for other active indolic compounds such as IAAld or IAA.