Adhesion as a Focus in Trichoderma-Root Interactions

Abstract

Fungal spores, germlings, and mycelia adhere to substrates, including host tissues. The adhesive forces depend on the substrate and on the adhesins, the fungal cell surface proteins. Attachment is often a prerequisite for the invasion of the host, hence its importance. Adhesion visibly precedes colonization of root surfaces and outer cortex layers, but little is known about the molecular details. We propose that by starting from what is already known from other fungi, including yeast and other filamentous pathogens and symbionts, the mechanism and function of Trichoderma adhesion will become accessible. There is a sequence, and perhaps functional, homology to other rhizosphere-competent Sordariomycetes. Specifically, Verticillium dahliae is a soil-borne pathogen that establishes itself in the xylem and causes destructive wilt disease. Metarhizium species are best-known as insect pathogens with biocontrol potential, but they also colonize roots. Verticillium orthologs of the yeast Flo8 transcription factor, Som1, and several other relevant genes are already under study for their roles in adhesion. Metarhizium encodes relevant adhesins. Trichoderma virens encodes homologs of Som1, as well as adhesin candidates. These genes should provide exciting leads toward the first step in the establishment of beneficial interactions with roots in the rhizosphere.

Keywords: Trichoderma; adhesin; adhesion; fungal; mutualism; rhizosphere; root; transcriptional.

Figure 1

Adhesion of germinating Trichoderma conidia. (A) Adherence of T. asperellum to cucumber roots in hydroponics, and role of a hydrophobin, TasHyd1 (image from [22], Molecular Plant Pathology, Wiley, open access). The roots were imaged 48 h post-inoculation: 1, non-inoculated control; 2, wild type; 3, TasHyd1 deletion mutant; 4, complemented strain. The large cells are root hairs. (B) Trichoderma virens germlings adhering in hydroponic culture. Left, germlings of T. virens adhering to maize roots in hydroponic culture: spores germinated 16 h to inoculation time, incubation with roots on a rotary shaker for 3 h, scale bar 10 mm; note decoration of root hairs with adhering germlings (arrow) (Horwitz, unpublished). Right, germlings accumulated on root hairs; scale bar approximately 250 μm (Taylor, unpublished).

Figure 2

Sample phylogenies of candidate Als group adhesins in Trichoderma species. Metarhizium anisopliae Mad1 and Mad2 [12] were used to search Trichoderma and a few selected ascomycete databases at JGI Mycocosm [42] or the NCBI protein database by BLASTP. The available, computer-generated annotations do not correspond to adhesins, and these genes have not been manually annotated. Annotations by simple numbers are JGI protein IDs, those beginning with letters are from NCBI, and VDAG_01815 is a V. dahliae database accession corresponding to JGI ID 6481. The species abbreviations are: Metani, Metarhizium anisopliae; Aspfu, Aspergillus fumigatus; Vda, Verticillium dahliae; Vlongi, Verticillium longisporum; Colgr, Colletotrichum graminearum; Acrchr, Acremonium chrysogenum; Canalb, Candida albicans; Trivi, Trichoderma virens; Triat, Trichoderma atroviride; Trire, Trichoderma reesei. The trees were generated at http://phylogeny.fr ([43], accessed on 30 March 2022; the numbers in red at each node are simulated bootstrap values). For the alignments shown below the trees, the sequences were aligned using CLUSTAL Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 25 March 2022). A portion of each of the two alignments, corresponding to part of a threonine-rich region identified in all these sequences by PROSITE (https://prosite.expasy.org/scanprosite/, accessed on 25 March 2022), is shown below each tree diagram (color coding indicates amino acid class; the symbols *: below alignments indicate identity/similarity). In the Mad1 phylogeny, A. chrysogenum and C. albicans are relatively distant, aligned with large gaps, and were excluded from the alignment shown. Likewise, a region of C. graminicola Mad2 that created a gap in the alignment of all the other sequences was removed manually before making the alignment shown. The trees were generated from the full alignments of all the complete sets of protein sequences.

Figure 3

Som1/Flo8 orthologs of several Trichoderma species. (A) Phylogeny generated at Phylogeny.fr: Phylogeny Analysis [43]. (B) Protein models. Predicted protein sequences and intron structures are from JGI Mycocosm (https://mycocosm.jgi.doe.gov/mycocosm/home, accessed on 19 December 2021; [42,44,45]). Red bars indicate coding sequence and blue bars, 5’ and 3’ untranslated regions. Species abbreviations in addition to those in Figure 2 are: Sc, Saccharomyces cerevisiae; Tasp, Trichoderma asperellum; FoL, Fusarium oxysporum f. sp. lycopersici 4287; Ashgo, Eremothecium (Ashbya) gossypii; Um, Ustilago maydis; Rs, Rhizoctonia solani; Colhi, Colletotrichum higginsianum. The numbers are JGI database protein IDs or NCBI accessions.

Figure 3

Som1/Flo8 orthologs of several Trichoderma species. (A) Phylogeny generated at Phylogeny.fr: Phylogeny Analysis [43]. (B) Protein models. Predicted protein sequences and intron structures are from JGI Mycocosm (https://mycocosm.jgi.doe.gov/mycocosm/home, accessed on 19 December 2021; [42,44,45]). Red bars indicate coding sequence and blue bars, 5’ and 3’ untranslated regions. Species abbreviations in addition to those in Figure 2 are: Sc, Saccharomyces cerevisiae; Tasp, Trichoderma asperellum; FoL, Fusarium oxysporum f. sp. lycopersici 4287; Ashgo, Eremothecium (Ashbya) gossypii; Um, Ustilago maydis; Rs, Rhizoctonia solani; Colhi, Colletotrichum higginsianum. The numbers are JGI database protein IDs or NCBI accessions.

Figure 4

Expression of TvSom1 and four adhesin candidates in interaction with maize roots. RNASeq data are shown for T. virens in interaction with maize in hydroponic cultures. Transcript abundance is plotted relative to mycelia in axenic culture, mean, and SEM of three independent samples, from data of [41]. Adhesin candidates are noted as “ad_” and the gene identifiers are JGI T. virens v2.0 protein ID numbers. ad_10277 is the GLEYA domain protein (see text), ad_261090 is a Mad2-like protein (Figure 2), and ad_172511 and ad_79838 are Fas1 and Wsc1 homologs, respectively. hpi, hours post-inoculation of Trichoderma germlings.

Figure 5

Network of V. dahliae transcription factors, which are involved in the regulation of adhesion. Black arrows indicate a transcriptional control which might be either direct or indirect. For example, the dashed line indicates regulation downstream of Som1; however, Vta2 is itself regulated by Som1. A possible Mad2-like adhesin (VDAG_01815) is a target of Mcm1 but not of VTA2. For further details see the main text.