Role of swollenin, an expansin-like protein from Trichoderma, in plant root colonization

Abstract

Swollenin, a protein first characterized in the saprophytic fungus Trichoderma reesei, contains an N-terminal carbohydrate-binding module family 1 domain (CBD) with cellulose-binding function and a C-terminal expansin-like domain. This protein was identified by liquid chromatography-mass spectrometry among many other cellulolytic proteins secreted in the coculture hydroponics medium of cucumber (Cucumis sativus) seedlings and Trichoderma asperellum, a well-known biocontrol agent and inducer of plant defense responses. The swollenin gene was isolated and its coding region was overexpressed in the same strain under the control of the constitutive pki1 promoter. Trichoderma transformants showed a remarkably increased ability to colonize cucumber roots within 6 h after inoculation. On the other hand, overexpressors of a truncated swollenin sequence bearing a 36-amino acid deletion of the CBD did not differ from the wild type, showing in vivo that this domain is necessary for full protein activity. Root colonization rates were reduced in transformants silenced in swollenin gene expression. A synthetic 36-mer swollenin CBD peptide was shown to be capable of stimulating local defense responses in cucumber roots and leaves and to afford local protection toward Botrytis cinerea and Pseudomonas syringae pv lachrymans infection. This indicates that the CBD domain might be recognized by the plant as a microbe-associated molecular pattern in the Trichoderma-plant interaction.

Figure 1.

Alignment of known swollenin sequences in fungi. Arrowhead indicates cleavage site of the signal peptide. Underlined regions indicate the CBD and pollen allergen 1 domains.

Figure 2.

Modulation of TasSwo expression in T. asperellum mycelium by different growth conditions. Gene induction was quantified by real-time RT-PCR, normalized versus the endogenous β-tubulin gene (see “Materials and Methods”). A, Total RNA was extracted from Trichoderma mycelium grown for 48 h in liquid SM medium with different carbon sources (1.5% Glc or 2% cellulose). B, Total RNA was extracted from Trichoderma mycelium wrapped around cucumber roots 12 h after inoculation. RNA extracted from mycelium of Trichoderma grown in hydroponic medium supplemented with 0.05% Glc was used as a control.

Figure 3.

Characterization of Trichoderma mutants. A, PCR screening of different mutants with primers detecting the transgene pki1-TasSwo and pki1-TasSwoΔCBD. pRL-swo and pRL-ΔCBD are positive plasmid controls. B, RT-PCR analysis of mutants expressing TasSwo (T1, T5, T6, T2) or TasSwoΔCBD (ΔT3, ΔT7, ΔT4) along with the wild type (WT) from RNA extracted from mycelia grown on 1.5% Glc.

Figure 4.

Root colonization by swollenin-overexpressing strains. Cucumber seedlings were infected with T. asperellum wild-type (WT), with TasSwo full-length (T1 and T2), and with TasSwo ΔCBD (T3 and T4) overexpression lines. Roots were detached 6 and 12 h postinoculation and washed. To quantify colonization, surface-sterilized roots were homogenized in water and plated on Trichoderma selective medium at 28°C and the number of fungal colonies (CFU) was counted. Fifty seedlings were tested in each treatment. Results are representative of three independent experiments. *, Significantly different from the WT group (P < 0.05; t test).

Figure 5.

Analysis of TasSwo RNAi lines PS1 and PS2. A, Real-time RT-PCR analysis of TasSwo RNAi mutants along with the wild type (WT) as control. Total RNA was extracted from T. asperellum mycelium grown for 48 h in SM liquid medium with 2% cellulose as carbon source. Results are representative of two independent experiments. B, Root colonization assay. Roots were detached 12 h postinoculation and washed. To quantify colonization, surface-sterilized roots were homogenized in water and plated on Trichoderma selective medium at 28°C and the number of fungal colonies (CFU) was counted. Fifty seedlings were tested in each treatment. Results are representative of three independent experiments. *, Significantly different from the WT group (P < 0.05; t test).

Figure 6.

Elicitation of plant defense genes by a synthetic 36-mer CBD peptide. A and B, Cucumber leaves were infiltrated with 0.002% Glc (mock) or 5 μm CBD. Twenty-four hours postinfiltration, total RNA was extracted from a pool of three leaves and used for real-time RT-PCR analysis of hpl, pal1, prx, chit, and β-gluc gene expression. The results are the average of three independent experiments. C, Cucumber seedlings were incubated in a 0.002% Glc solution (mock) or in 10 μm CBD for 24 h. Total RNA was then extracted from a pool of five roots and used for real-time RT-PCR analysis. The results are the average of two independent experiments.

Figure 7.

Protection against fungal and bacterial plant pathogens by CBD. A, Cucumber leaves were infiltrated with 100 mL of 0.002% Glc (mock) or 5 μm CBD and were inoculated after 24 h with B. cinerea mycelium (see “Materials and Methods”). Disease symptoms are visible 3 to 4 d after inoculations. B, Disease index, from 0 (no symptom) to 3 (severe lesion), was assessed according to the disease symptoms of 10 leaves for each treatment. The results are the average of three independent experiments. C, Psl multiplication in cotyledons of cucumber seedlings treated with 5 μm CBD or 0.002% Glc (control) 24 h prior to bacteria challenge. Proliferation of bacteria in plant leaves was assayed 2 d after inoculation by monitoring bacterial growth from leaf extracts. Each bar represents the mean of 45 replicates that was obtained in five independent experiments. *, Significantly different from the mock treatment (P < 0.05; t test).