Ecologically Different Fungi Affect Arabidopsis Development: Contribution of Soluble and Volatile Compounds

<div><p>Plant growth and development can be influenced by mutualistic and non-mutualistic microorganisms. We investigated the ability of the ericoid endomycorrhizal fungus <i>Oidiodendron maius</i> to influence growth and development of the non-host plant <i>Arabidopsis thaliana</i>. Different experimental setups (non-compartmented and compartmented co-culture plates) were used to investigate the influence of both soluble and volatile fungal molecules on the plant phenotype. <i>O</i>. <i>maius</i> promoted growth of <i>A</i>. <i>thaliana</i> in all experimental setups. In addition, a peculiar clumped root phenotype, characterized by shortening of the primary root and by an increase of lateral root length and number, was observed in <i>A</i>. <i>thaliana</i> only in the non-compartmented plates, suggesting that soluble diffusible molecules are responsible for this root morphology. Fungal auxin does not seem to be involved in plant growth promotion and in the clumped root phenotype because co-cultivation with <i>O</i>. <i>maius</i> did not change auxin accumulation in plant tissues, as assessed in plants carrying the DR5::GUS reporter construct. In addition, no correlation between the amount of fungal auxin produced and the plant root phenotype was observed in an <i>O</i>. <i>maius</i> mutant unable to induce the clumped root phenotype in <i>A</i>. <i>thaliana</i>. Addition of active charcoal, a VOC absorbant, in the compartmented plates did not modify plant growth promotion, suggesting that VOCs are not involved in this phenomenon. The low VOCs emission measured for <i>O</i>. <i>maius</i> further corroborated this hypothesis. By contrast, the addition of CO₂ traps in the compartmented plates drastically reduced plant growth, suggesting involvement of fungal CO₂ in plant growth promotion. Other mycorrhizal fungi, as well as a saprotrophic and a pathogenic fungus, were also tested with the same experimental setups. In the non-compartmented plates, most fungi promoted <i>A</i>. <i>thaliana</i> growth and some could induce the clumped root phenotype. In the compartmented plate experiments, a general induction of plant growth was observed for most other fungi, especially those producing higher biomass, further strengthening the role of a nonspecific mechanism, such as CO₂ emission.</p></div

The small secreted protein OmSSP1 is up-regulated in ericoid mycorrhiza and is involved in symbiosis

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The Hydrophobin-Like OmSSP1 May Be an Effector in the Ericoid Mycorrhizal Symbiosis

Mutualistic and pathogenic plant-colonizing fungi use effector molecules to manipulate the host cell metabolism to allow plant tissue invasion. Some small secreted proteins (SSPs) have been identified as fungal effectors in both ectomycorrhizal and arbuscular mycorrhizal fungi, but it is currently unknown whether SSPs also play a role as effectors in other mycorrhizal associations. Ericoid mycorrhiza is a specific endomycorrhizal type that involves symbiotic fungi mostly belonging to the Leotiomycetes (Ascomycetes) and plants in the family Ericaceae. Genomic and RNASeq data from the ericoid mycorrhizal fungus Oidiodendron maius led to the identification of several symbiosis-upregulated genes encoding putative SSPs. OmSSP1, the most highly symbiosis up-regulated SSP, was found to share some features with fungal hydrophobins, even though it lacks the Pfam hydrophobin domain. Sequence alignment with other hydrophobins and hydrophobin-like fungal proteins placed OmSSP1 within Class I hydrophobins. However, the predicted features of OmSSP1 may suggest a distinct type of hydrophobin-like proteins. The presence of a predicted signal peptide and a yeast-based signal sequence trap assay demonstrate that OmSSP1 is secreted. OmSSP1 null-mutants showed a reduced capacity to form ericoid mycorrhiza with Vaccinium myrtillus roots, suggesting a role as effectors in the ericoid mycorrhizal interaction

<i>O</i>. <i>maius—A</i>. <i>thaliana</i> co-cultivation experiments in the tripartite plate system.

<p>(a) Control plants and plant-fungus co-culture 15 days after inoculation; (b) same as in (a) but plates were added with a VOC trap (activated charcoal, AC) in the third compartment; (c) same as in (a) but plates were added with a CO₂ trap [Ba(OH)_2*8H₂O, B] together with two dental rolls in the third compartment; (d) plant biomass measurements (roots—grey bars—and aboveground portions—open bars) in the presence/absence of the fungus and of the trap compounds. Note the strong plant biomass increase in the presence of <i>O</i>. <i>maius</i> in all the conditions tested. Bars represent the mean ±SD, n = 5 (each biological replicate represents the total biomass of 3 <i>A</i>. <i>thaliana</i> seedlings grown in an individual plate). Statistically significant differences (P<0.05) among treatments are indicated by different letters above the bars.</p

<i>A</i>. <i>thaliana</i> development in the presence of <i>O</i>. <i>maius</i> WT (Om) and of three <i>O</i>. <i>maius</i> mutants (OmΔGOGAT; OmΔMFS; OmΔSOD).

<p>(a) Control plants (C) and plant-fungus co-cultures 30 days after inoculation (all pictures were taken at the same magnification) (b) Measurement of auxin quantity released in the culture medium by <i>O</i>. <i>maius</i> WT and by the three <i>O</i>. <i>maius</i> mutants, using the Salkowski reaction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168236#pone.0168236.ref046" target="_blank">46</a>]. Auxin quantity measured was normalized to the mycelium biomass. Bars represent the mean ±SD, n = 3 (each biological replicate represents the total biomass of 5 <i>A</i>. <i>thaliana</i> seedlings grown in an individual plate). Statistically significant differences (P<0.05) among treatments are indicated by different letters above the bars.</p

Analysis of <i>A</i>. <i>thaliana</i> root development.

<p>Average quantification of root parameters in 20 individual <i>A</i>. <i>thaliana</i> plants grown alone or co-cultured for 9 days with the <i>O</i>. <i>maius</i> WT strain (Om) or the <i>O</i>. <i>maius</i> GOGAT mutant (OmΔGOGAT) is plotted into charts. Primary (PR), secondary (SRs) and tertiary roots (TRs) were counted and measured using the ImageJ plug in SmartRoot software. A diagrammatic representation of <i>A</i>. <i>thaliana</i> root development in the different conditions tested is also shown.</p

VOC emission profiles of the <i>O</i>. <i>maius</i> WT and of the <i>O</i>. <i>maius</i> GOGAT mutant.

<p>VOCs were collected in the headspace of culture plates 15 (open and hatched bars) and 30 (black and dotted bars) days after inoculation. Bars represent the mean ±SD as pmol cm^-2 h^-1, n = 6. (OCT) 1-octen-3-ol; (CAD) epsilon-cadinene; (PHE) phenol,2,4-bis(1,1-dimethylethyl); (GER) germacrene D.</p

<i>A</i>. <i>thaliana</i> development in the presence of different fungi in the bipartite plate system.

<p>(a) Control plants and plant-fungus co-cultures 15 days after inoculation; (b) plant biomass measurements (roots—grey bars—and aboveground portions—open bars) in the presence/absence of fungi. Note the strong plant biomass increase in the presence of some of the fungi tested. Bars represent the mean ±SD, n = 5 (each biological replicate represents the total biomass of 3 <i>A</i>. <i>thaliana</i> seedlings grown in an individual plate). Statistically significant differences (P<0.05) among treatments are indicated by different letters above the bars. Om, <i>Oidiodendron maius</i>; Mb, <i>Meliniomyces bicolor</i>; Mv, <i>Meliniomyces variabilis</i>; Re, <i>Rhizoscyphus ericae</i>; Lb, <i>Laccaria bicolor</i>; Cg, <i>Cenococcum geophilum</i>; Sl, <i>Suillus luteus</i>; Tc, <i>Tulasnella calospora</i>; Tv, <i>Trametes versicolor</i>; Ch, <i>Cladosporium herbarum</i>.</p

Data_Sheet_1_The Hydrophobin-Like OmSSP1 May Be an Effector in the Ericoid Mycorrhizal Symbiosis.docx

<p>Mutualistic and pathogenic plant-colonizing fungi use effector molecules to manipulate the host cell metabolism to allow plant tissue invasion. Some small secreted proteins (SSPs) have been identified as fungal effectors in both ectomycorrhizal and arbuscular mycorrhizal fungi, but it is currently unknown whether SSPs also play a role as effectors in other mycorrhizal associations. Ericoid mycorrhiza is a specific endomycorrhizal type that involves symbiotic fungi mostly belonging to the Leotiomycetes (Ascomycetes) and plants in the family Ericaceae. Genomic and RNASeq data from the ericoid mycorrhizal fungus Oidiodendron maius led to the identification of several symbiosis-upregulated genes encoding putative SSPs. OmSSP1, the most highly symbiosis up-regulated SSP, was found to share some features with fungal hydrophobins, even though it lacks the Pfam hydrophobin domain. Sequence alignment with other hydrophobins and hydrophobin-like fungal proteins placed OmSSP1 within Class I hydrophobins. However, the predicted features of OmSSP1 may suggest a distinct type of hydrophobin-like proteins. The presence of a predicted signal peptide and a yeast-based signal sequence trap assay demonstrate that OmSSP1 is secreted. OmSSP1 null-mutants showed a reduced capacity to form ericoid mycorrhiza with Vaccinium myrtillus roots, suggesting a role as effectors in the ericoid mycorrhizal interaction.</p

<i>A</i>. <i>thaliana</i> development in the presence of <i>O</i>. <i>maius</i> and of nine other fungi.

<p>(a) Control plants (C) and plant-fungus co-cultures 30 days after inoculation; (b) plant biomass measurements (roots—grey bars—and aboveground portions—open bars) in the presence/absence of fungi. Note the strong plant biomass increase in the presence of some of the fungi tested. Bars represent the mean ±SD, n = 5 (each biological replicate represents the total biomass of 5 <i>A</i>. <i>thaliana</i> seedlings grown in an individual plate). Statistically significant differences (P<0.05) among treatments are indicated by different letters above the bars. Om, <i>Oidiodendron maius</i>; Mb, <i>Meliniomyces bicolor</i>; Mv, <i>Meliniomyces variabilis</i>; Re, <i>Rhizoscyphus ericae</i>; Lb, <i>Laccaria bicolor</i>; Sl, <i>Suillus luteus</i>; Cg, <i>Cenococcum geophilum</i>; Tc, <i>Tulasnella calospora</i>; Ch, <i>Cladosporium herbarum</i>; Tv, <i>Trametes versicolor</i>.</p