Identification of effector-like proteins in Trichoderma spp. and role of a hydrophobin in the plant-fungus interaction and mycoparasitism

Abstract

Background: Trichoderma spp. can establish beneficial interactions with plants by promoting plant growth and defense systems, as well as, antagonizing fungal phytopathogens in mycoparasitic interactions. Such interactions depend on signal exchange between both participants and can be mediated by effector proteins that alter the host cell structure and function, allowing the establishment of the relationship. The main purpose of this work was to identify, using computational methods, candidates of effector proteins from T. virens, T. atroviride and T. reesei, validate the expression of some of the genes during a beneficial interaction and mycoparasitism and to define the biological function for one of them.

Results: We defined a catalogue of putative effector proteins from T. virens, T. atroviride and T. reesei. We further validated the expression of 16 genes encoding putative effector proteins from T. virens and T. atroviride during the interaction with the plant Arabidopsis thaliana, and with two anastomosis groups of the phytopathogenic fungus Rhizoctonia solani. We found genes which transcript levels are modified in response to the presence of both plant fungi, as well as genes that respond only to either a plant or a fungal host. Further, we show that overexpression of the gene tvhydii1, a Class II hydrophobin family member, enhances the antagonistic activity of T. virens against R. solani AG2. Further, deletion of tvhydii1 results in reduced colonization of plant roots, while its overexpression increases it.

Conclusions: Our results show that Trichoderma is able to respond in different ways to the presence of a plant or a fungal host, and it can even distinguish between different strains of fungi of a given species. The putative effector proteins identified here may play roles in preventing perception of the fungus by its hosts, favoring host colonization or protecting it from the host's defense response. Finally, the novel effector protein TVHYDII1 plays a role in plant root colonization by T, virens, and participates in its antagonistic activity against R. solani.

Keywords: Effector; Hydrophobin; Mycoparasitism; Plant-fungus interaction; Trichoderma.

Fig. 1

Bioinformatic pipeline for the identification of effector proteins in Trichoderma spp. The pipeline is composed of five major steps. Step 1 (box 1), extracellular proteins where predicted using the Secretor algorithm (designed and constructed for this work). Step 2 (box 2), functional annotation of the extracellular proteins, using Pfam database and InterPro pipelines. Step 3 (box 3) individual extracellular proteins where searched for translocation and nuclear localization signal motifs. Step 4 (box 4), individual extracellular proteins where annotated using current knowledge of effector properties. Step 5 (box 5), ranking and classification of the effector candidates based on the different effector properties matched by each protein. Tv, Trichoderma virens; Ta, Trichoderma atroviride; Tr, Trichoderma reseei

Fig. 2

Expression of putative effector encoding genes from T. virens in interaction with A. thaliana. a T. virens – A. thaliana culture plates before contact (BC), at initial contact (C) and at overgrowth (OG) stages. b–f, expression level of the possible effector coding genes selected from T. virens at BC, C and OG stages of interaction with Arabidopsis thaliana. 2^(−ΔΔCt) values represent the change in the expression level of each gene compared to the control condition (fungus growing alone), which has a designated value of 1 (dotted line). Data were analyzed with a two-way ANOVA and a Bonferroni post hoc test; *** p < 0.001. Values represent means of three replicates, bars indicate standard deviation

Fig. 3

Expression of putative effector encoding genes from T. atroviride in interaction with A. thaliana. a T. atroviride – A. thaliana culture plates before contact (BC), at initial contact (C) and at overgrowth (OG) stages. b–f, expression level of the possible effector coding genes selected from T. atroviride at BC, C and OG stages of interaction with Arabidopsis thaliana. 2^(−ΔΔCt) values represent the change in the expression level of each gene compared to the control condition (fungus growing alone), which has a designated value of 1 (dotted line). Data were analyzed with a two-way ANOVA and a Bonferroni post hoc test; *** p < 0.001. Values represent means of three replicates, bars indicate standard deviation

Fig. 4

Expression level of putative effector encoding genes from Trichoderma in confrontation with Rhizoctonia solani AG5. a T. atroviride – R. solani AG5 culture plates before contact (BC), at initial contact (C) and at overgrowth (OG) stages. b – e, expression level of the selected genes tvlysm1, tvsep3, tvhydii1 and tvmp1 from T. virens. f-g, expression level of the selected genes talysm1, tacfem1 and epl2 from T. atroviride. BC, before contact between both fungi; C, initial contact of the fungi; OG, growth of Trichoderma over R. solani AG5. 2^(−ΔΔCt) values represent the change in the expression level of each gene compared to the control condition (fungus growing alone), which has a designated value of 1 (dotted line). Data were analyzed with a two-way ANOVA and a Bonferroni post hoc test; * p < 0.05; ** p < 0.01; *** p < 0.001. Values represent means of three replicates, bars indicate standard deviation

Fig. 5

Expression level of putative effector encoding genes from Trichoderma in confrontation with Rhizoctonia solani AG2. a – d, expression level of the selected genes tvlysm1, tvsep3, tvhydii1 and tvmp1 from T. virens. e–g, expression level of the selected genes talysm1, tacfem1 and epl2 from T. atroviride. BC, before contact between both fungi; C, initial contact of the fungi; OG, growth of Trichoderma over R. solani AG2. 2^(−ΔΔCt) values represent the change in the expression level of each gene compared to the control condition (fungus growing alone), which has a designated value of 1 (dotted line). Data were analyzed with a two-way ANOVA and a Bonferroni post hoc test; * p < 0.05; ** p < 0.01; *** p < 0.001. Values represent means of three replicates, bars indicate standard deviation

Fig. 6

tvhydii1 knockout and overexpressing strains. a confirmation of the tvhydii1 gene deletion; lane 1, 5’ end of the deletion cassette inserted at the tvhydii1 locus; lane 2, tvhydii1 wild type amplification as control; lane 3, tvhydii1 amplification in the knockout strains; lane 4, 3’ end of the deletion cassette inserted at the tvhydii1 locus. b semi-quantitative RT-PCR of the knockout and overexpressing strains; RT-PCR reactions were carried out using gpd gene as amplification control for tvhydii1 gene; c hydrophobicity assay, a 20 μL drop of distilled water was placed over plugs of 72 h mycelia of each T. virens strain

Fig. 7

Mycoparasitic characterization of tvhydii1 strains. Confrontation plates between R. solani AG2 and each tvhydii1 strain at 5 and 7dpi. Front, photographs of the upper part of the plates; Back, photographs of the bottom part of the plates. Plugs of 72 h mycelia were placed in opposite parts of potato dextrose agar plates, confronting the selected tvhydii1 strain with the phytopathogen. Plates were placed at 28 °C in total darkness for 14 days

Fig. 8

Root colonization assay of tvhydii1 strains. Relative quantification of sm1 amplification in tomato roots colonized by T. virens 29.8, Δtvhydii1 T2.1, Δtvhydii1 T2.3, tvhydii1OE T2 and tvhydii1OE T5. 2^(−ΔΔCt), values represent the amplification level of sm1 gene relative to the amplification level of the expressed gene of the plant DNA, compared to the control condition (tomato DNA). T. virens 29.8 and S. lycopersicum DNA were used as amplification controls. Data were analyzed with a one-way ANOVA and a Bonferroni post hoc test; ** p < 0.01; *** p < 0.001. Values represent means of three replicates, bars indicate standard deviation