The search for effector symbiosis-related proteins in the ericoid endomycorrhizal fungus Oidiodendron maius

PI  : Elena Martino (UMR 1136 « Interactions Arbres/Micro-organismes » IAM  et Department of Life Sciences and Systems Biology, University of Turin, Italy

Co-applicants :
C. Veneault-Fourrey (UMR 1136, IaM, INRA-Lorraine Centre of Nancy)
A. Kohler (UMR 1136, IaM, INRA-Lorraine Centre of Nancy)

Collaboration :
S. Daghino (Department of Life Sciences and Systems Biology, University of Turin, Italy)
S. Perotto (Department of Life Sciences and Systems Biology, University of Turin, Italy)

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Context — Mycorrhizae are a fascinating world and an ecologically relevant system; over 90% of terrestrial plants have established interactions with mycorrhizal fungi, beginning at least 450 million years ago. Therefore, research on mycorrhizae is crucial for understanding the functioning of forest ecosystems. Ericoid mycorrhizal fungi (ERM) are soil-born fungi forming endomycorrhizae with plants within the family Ericaceae (e.g. blueberry). In addition to their economic and gastronomic interest, blueberries are important underbrush elements and special players in the carbon cycle. Mycorrhizal fungi form intimate intracellular contacts with living plant cells, they need therefore to manipulate host defences and metabolism. Looking at the biology of the system, to understand how two eukaryotes interact and communicate, remains very challenging. Studies on genes⁄functions involved in pathogenicity and symbiosis have highlighted a particular class of effectors corresponding to small secreted proteins (SSPs). While effector secreted proteins have been reported for AM and ECM fungi (Kloppholz et al. 2011; Plett et al. 2011), no similar studies have been reported for ERM fungi. Results of the first year for the EffectER project, showed that Vaccinium myrtillus plants colonized by Oidiodendron maius doubled their biomass and transcriptomic analyses revealed that 20% of O. maius SSPs are overexpressed in mycorrhizal roots.

Objectives —  The second year of the project considered the influence of a specific heavy metal – cadmium – on the ericoid fungus alone (free living mycelium) or in association with its host plant. Specific aims of the second year were: a) to analyse RNAseq available data obtained by extracting RNA from the O. maius free living mycelium and from V. myrtillus mycorrhizal and non-mycorrhizal roots searching for Cd-regulated genes in the three different conditions; b) to functionally characterized the most up-regulated genes in the symbiosis condition identified during the first year of the project

Approaches — Transcriptomic data were analyzed to find Cd regulated genes in the fungus alone, in the symbiotic condition and in the plant non-inoculated roots. Supplementary bioinformatics analyses, the yeast invertase assay and the transient expression of GFP fusions in tobacco leaves were also performed.

Key results —

• RNAseq data obtained by extracting RNA from the maius free living mycelium (FLM) and from V. myrtillus mycorrhizal (MYC) and non-mycorrhizal roots (C) were analysed searching for Cd-regulated genes. The Cd concentration used caused the up-regulation of 19 genes in the FLM. Among them there are: 6 SSPs, 4 oxidoreductases, 2 hydrolases and 7 genes with unknown functions. 27 fungal up-regulated genes were identified when comparing mycorrhizal roots treated with Cd with mycorrhizal untreated roots. Among them are 2 SSPs, 4 transporters, 10 oxidoreductases. Interestingly a significant increase of the up-regulated genes belonging to the transporters class was observed when comparing the Cd treated MYC condition to the Cd treated FLM condition.
• Cd-regulated plant genes in the Non-MYC and MYC conditions were analysed. The Cd-treated MYC condition contains the higher number of up-regulated transporters belonging to different families. In particular among the uniquely MYC Cd-induced genes the higher percentage is represented by Zn/Cu and SO₄-transporters.
• Transcriptomic data obtained for the FLM and the MYC conditions of maius associated with V. myrtillus plants revealed that among the 995 symbiosis up-regulated genes 90 correspond to SSPs. The most highly symbiosis up-regulated SSP (called O. maius SSPb) was chosen for further characterization. SSPb gene structure is composed of 354 nucleotides (2 exons and 1 intron) and this gene is located on the scaffold 14 of the O. maius genome in one single copy. The gene codes for a protein of 93 aa (9.24 KDa) rich in glycine (16.1%) and leucine (12.9%) and having 8 characteristic cysteine residues. No known domains could be found neither a functional classification. It possesses a Signal Peptide (SP) and a low complexity region. 3 orthologous genes were found for SSPb, they belong to the saprotrophic fungi Neurospora crassa and Thielavia terrestris and are annotated as hydrophobins. Blast alignment revels that O. maius SSPb shared similarities with others fungal hydrophobins. The Yeast-Invertase assay suggested that SSPb is secreted, although the test needs additional controls. An ectopic expression experiment in tobacco leaves suggested a membrane-cytoplasmic localization for the full length protein SSPb-GFP with no evidence of massive aspecific nuclear localization. Fluorescence localized along the cells and nuclear boundary and at some cytoplasmic spikes. The possible membrane-cytoplasmic localization was also corroborated by the fact that the fluorescence moves to the inner part of the cells after plasmolysis induction.

Main findings —

• SSPs are mostly studied in the contest of microorganisms interaction with plants. SSPs upregulation by Cd stress represents an interesting observation and opens up new perspectives in the study of SSPs functions, as also reported by Dorè and Perraud (2015) who suggested that the SSPs produced by the ECM fungus Hebeloma cylindrosporum may be involved in environmental stress response.
• An increase of transporters coding genes was observed for the mycorrhizal condition in the presence of Cd. In particular, among the uniquely MYC Cd-induced transporters an increase of Zn/Cu transporters belonging to the ZIP family was observed. Cd up-regulated SO₄– transporters also increase in the mycorrhizal condition.
• In ECM fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins: O. maius SSPb is highly expressed in mycorrhizal tissues and shares similarities with others fungal hydrophobins. Anyway, our preliminary localization data do not suggest a cell wall localization for SSPb.

Future perspectives —

• To better investigate the roles of SSPs and transporters in response to Cd in the MYC condition.
• To continue the functional characterization of the O. maius SSPb: a knock-out experiment is currently ongoing