A 2-component system is involved in the early stages of the Pisolithus tinctorius-Pinus greggii symbiosis.

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.

Detection of a histidine kinase mRNA in extraradical mycelium of Pisolithus tinctorius induced by the plant metabolites.

The aim of this study was to test the effect of acetosyringone (AS) on the accumulation of the histidine kinase coding mRNA, using in vitro propagated Pisolithus tinctorius. In the precontact phase of the ectomycorrhizal symbiosis, it is hypothesized that the plants releases chemicals, which are sensed by the fungal mycelium and in turn trigger the start of the symbiosis. The nature of such molecules is largely unknown; however, plant metabolite and their structural analogues have been widely used to induce infection in different microbe-plant interactions. A histidine kinase in Agrobacterium tumefaciens was activated by AS in the first step during the infection of dicotyledonous plants. A conserved gene fragment from the ectomycorrhizal fungus Pisolithus tinctorius was cloned using degenerate primers of conserved regions characteristic of the histidine kinase gene family. Then, the accumulation of this transcript in fungi incubated with AS was analyzed. RT-PCR and in situ hybridization suggest that this mRNA is synthesized in the presence of this plant analog in AS-treated mycelia. The findings presented here suggest a role of a histidine kinase involved in the early stages of ectomycorrhizal differentiation.

Stable genetic transformation of the ectomycorrhizal fungus Pisolithus tinctorius.

In the present work the genetic transformation and the expression of gene markers in transgenic Pisolithus tinctorius are reported. The ectomycorrhizae are facultative symbionts of plant roots, which are capable of affording mineral nutrients to its co-host in exchange of fixed carbon. Given the importance of this association (more than 80% of gymnosperms are associated with these fungi), its study from both basic and applied viewpoints is relevant. We have transformed this fungus with reporter genes and analyzed their expression in its saprophytic state. Genetic transformation was performed by microprojectile bombardment and Agrobacterium-mediated transformation. This last method proved to be the more efficient. Southern analysis of biolistic-transformed fungi revealed the random integration of the transgene into the genome. The accumulation of the transcript of the reporter gene was demonstrated by RT-PCR. The visualization of GFP-associated fluorescence in saprophytic mycelia confirmed the expression of the reporter gene. This is the first report on the stable transformation and expression of GFP in the ectomycorrhizal fungus P. tinctorius.

Efficient transformation of Cellulomonas flavigena by electroporation and conjugation with Bacillus thuringiensis.

The conjugative self-transmissible plasmid pHT73, harbored in Bacillus thuringiensis var. kurstaki, was demonstrated to be transferred to Cellulomonas flavigena, a cellulolytic bacterium. Both conjugation and transformation procedures yielded resistant colonies; however, chromosomal integration was observed only when bacterial conjugation occurred. The efficiency of conjugation was 10% of recipient strain, which is considered a very efficient process. When the plasmid pHT73 was introduced by transformation, erythromycin-resistant cells contained the plasmid as an episome with no arrangements, as assayed by Southern blot analysis. In contrast, conjugated-resistant cells harbor the plasmid integrated into the chromosome. These data suggest a common mechanism of cell communication between nonrelated bacterial species with similar ecological habitats, and also that both electroporation and conjugation can be used to transform C. flavigena efficiently.