New advances on ectomycorrhizal establishment : learning from transcriptome and metabolome analysis

Ectomycorrhiza formation encompasses changes in soil fungi and vascular plants roots. Host plants metabolites released to the rhizosphere trigger spore germination, hyphae growth towards the root inducing mycorrhiza formation. Plant and fungal genetic switches necessary for ECM development remain unidentified and little is known on the initial contact phase. The model system studied is the mycorrhizal association Castanea sativa / Pisolithus tinctorius and Quercus suber / Pisolithus tinctorius. The mycorrhiza formation followed two different methods. The association C. sativa / P. tinctorius mycorrhization was induced in liquid medium as previously described (Sebastiana et al. 2009), while that of Q. suber / P. tinctorius was induced as follows:- (1) Pure culture of P. tinctorius was obtained from a sporocarp collected at the base of a cork oak tree (Minas de Aljustrel, Alentejo), development of mycorrhizal inoculum for maximum biomass production in a carrier substrate (peatmoss and vermiculite)inoculation of Q. suber seedlings in the greenhouse by transferring the roots to inoculated potting substrate. 4 months after P. tinctorius inoculation, 100% of the plants were mycorrhized with an average mycorrhized root length of 46%. Metabolome profiling of C. sativa / P. tinctorius revealed metabolites crucial for mycorrhiza formation, at the plant level:- stress and defence related amino acids (proline, alanine and glycine), cell wall synthesis and degradation related metabolites; saccharides (glucose, fructose and sucrose); galacturonic acid (indicator of cell wall destruction); aromatic amino acids and signalling related metabolites (myo-inositol, polyamines, galactinol) and at the fungi level:- trehalose-fungi (marker of fungi presence); gluconic acid (promoter of ectomycorrhizae formation); glutamine (marker of mantle and Hartig net formation). Transcriptome profiling revealed, up regulation of genes encoding for stress and defence response proteins (cystatin, cystatin-like protein, defensin and a universal stress protein). Early contact with the fungal mycelium altered expression of genes putatively involved in cellular processes:- signal transduction and communication (receptor kinase-related protein), protein fate (papain-like cystein proteinase) and water transport facilitation (water channel MipK protein). The transcriptome of mycorrhizal cork oak association revealed up-regulation of ACX1 (Acyl-CoA Oxidase), CYP74 (Allene oxide synthase), Allene Oxide Synthase, OPR3 (12-Oxophytodienoate reductase 3), OPCL1 (4-Coumarate-CoA Ligase), Fatty Acid Desaturase, Ethylene Transcription Factor, Hydroxyacyl-CoA Dehydrogenase, Acyl-CoA Oxidase, Omega-3 Fatty Acid Desaturase, Catalase, and of Long Chain Acyl-CoA Synthetase 4. Taking together the metabolome and transcriptome analysis in Chestnut and cork-oak mycorrhizal associations there is a strong evidence of an important signaling pathway. Results on the over expression of genes related to Jasmonic acid and proline biosynthesis are discussed. Expression profiling in early C. sativa / P. tinctorius contact revealed changes in gene expression occurring few hours after contact, long before developing a functional mycorrhiza. Induction of genes involved on stress and defense suggests a quick reaction of host plant to the mycobiont presence, eliciting a defence programme similar to that described for pathogenic interactions. Repression of genes related to water stress accounts for a water stress relief resulting from the initial contact with ectomycorrhizal fungus