Project description:Ectomycorrhizal symbiosis results in profound morphological and physiological modifications in both plant and fungus. This in turn is the product of differential gene expression in both co-symbionts, giving rise to specialized cell types capable of performing novel functions. During the precolonization stage, chemical signals from root exudates are sensed by the ectomycorrizal fungus, and vice versa, which are in principle responsible for the observed change in the developmental symbionts program. Little is known about the molecular mechanisms involved in the signaling and recognition between ectomycorrhizal fungi and their host plants. In the present work, we characterized a novel lactone, termed pinelactone, and identified a gene encoding for a histidine kinase in Pisolithus tictorius, which function is proposed to be the perception of the aforementioned metabolites. In this study, the use of closantel, a specific inhibitor of histidine kinase phosphorylation, affected the capacity for fungal colonization in the symbiosis between Pisolithus tinctorius and Pinus greggii, indicating that a 2-component system (TCS) may operate in the early events of plant-fungus interaction. Indeed, the metabolites induced the accumulation of Pisolithus tinctorius mRNA for a putative histidine kinase (termed Pthik1). Of note, Pthik1 was able to partially complement a S. cerevisiae histidine kinase mutant, demonstrating its role in the response to the presence of the aforementioned metabolites. Our results indicate a role of a 2-component pathway in the early stages of ectomycorrhizal symbiosis before colonization. Furthermore, a novel lactone from Pinus greggii root exudates may activate a signal transduction pathway that contributes to the establishment of the ectomycorrhizal symbiosis.
Project description:PtSRR1 EST was previously identified in the first hours of Pisolithus tinctorius and Castanea sativa interaction. QRT-PCR confirmed PtSRR1 early expression and in silico preliminary translated peptide analysis indicated a strong probability that PtSRR1 be a transmembrane protein. These data stimulate the PtSRR1 gene research during ectomycorrhiza formation.
Project description:Ectomycorrhiza is a complex association of several types of plant and fungal cells. Differentiation of symbiotic structures is correlated with large changes in mRNA synthesis, leading to novel protein patterns. Quantification of up- and down-regulated specific transcripts is complicated by the intermingling of root and hyphal components. Determination of steady-state levels of symbiosis-regulated mRNA requires a normalization to the housekeeping RNA content of each partner. In this study, the usefulness of the internal transcribed spacer (ITS)-5.8S ribosomal DNAs (rDNAs) as molecular markers of the root colonization by fungal mycelium was assayed. The rDNA ITSs of Pisolithus tinctorius and Eucalyptus globulus were cloned by PCR amplification, and their sequences were determined. They contained the 5.8S rDNAs, and these two probes did not cross-hybridize. Steady-state levels of the ITS-5.8S rRNAs in the vegetative mycelium, in the noninfected root, and in ectomycorrhizas of E. globulus-P. tinctorius 441 were estimated at different stages of development. Colonization of roots by the mycelium provoked a large decrease in the proportion of root rRNAs. At the end of mycorrhiza formation, about 80% of the ectomycorrhizal RNA belonged to the mycobiont. The ITS-5.8S can be used as a specific probe for the estimation of fungal or plant rRNA in the symbiotic tissues and to determine whether an mRNA is down- or up-regulated in ectomycorrhiza.