Characterization of organic nitrogen transport in the ectomycorrhizal fungus Hebeloma cylindrosporum

Ektomykorrhizen haben in borealen Nadelwäldern und temperierten Laubwäldern der Nordhemisphäre eine entscheidene Aufgabe in der Versorgung ihrer pflanzlichen Partner mit Stickstoff, der wichtigste wachstumslimitierende Nährstoff. In diesen Waldböden ist Stickstoff hauptsächlich in organischen Verbindungen vorhanden, welche nicht für die Pflanze, aber für den Ektomykorrhizapilz verwertbar sind. Die mit der Pflanze in Symbiose lebenden Pilze können mit Hilfe von extrazellulären Proteinasen Proteine in diesen Böden spalten. Dies ermöglicht dem Pilz organischen Stickstoff in Form von freien Aminosäuren und Peptiden aufzunehmen, zu assimilieren und schliesslich (zum Teil) an die Pflanze abzugeben. Da der Ektomykorrhizapilz einen wichtigen Beitrag zur Stickstoffversorgung für die mit ihm assoziierten Pflanze leistet, ist es von Bedeutung, die Aufnahmemechanismen von organischen Stickstoff vom Boden in den Pilz (und schliesslich Transport in die Pflanze) und deren Regulation zu verstehen. Um Transporter zu identifizieren, die an der Aufnahme von organischen Stickstoffverbindungen aus dem Boden in die Pilzhyphen bzw. am Transport vom Pilz in die Pflanze, beteiligt sind wurde eine cDNA Bibliothek vom Myzel des Ektomykorrhizapilzes Hebeloma cylindrosporum hergestellt. In dieser Arbeit wurde die Qualität dieser Bibliothek getestet, indem ca. 500 ESTs sequenziert wurden und damit eine Sequenzdatenbank für den Modellorganismus H. cylindrosporum hergestellt (www.uni-tuebingen.de/plantphys /hebeloma/index.html). Von dieser cDNA Bibliothek wurde ein Gen, das für einen Aminosäuretransporter (HcGAP1) kodiert isoliert und charakterisiert. Zusätzlich wurden zwei Peptidtransporter (HcPTR2A, -B) charakterisiert. Die Analyse dieser Transporter durch Aufnahmeexperimenten zeigt, das sie am Import von organischen Stickstoffverbindungen beteiligt sind. Weiterhin konnte durch Expressionsstudien gezeigt werden, dass diese Transporter durch verschiedene Stickstoffquellen reguliert werden. Da extrazelluläre Proteinasen eine wichtige Rolle für die Verfügbarkeit von organischen Stickstoff für den Pilz bzw die Pflanze spielen, wurde die Proteaseaktivität von Hebeloma getestet und ein Gen das möglicherweise für eine Proteinase kodiert isoliert. Schliesslich wurde eine Methode zur Transformation von Hebeloma via Agrobakterium tumefaciens etabliert.Ectomycorrhizal trees dominate boreal and temperate forest ecosystems in which nitrogen is generally accepted to be the most important growth-limiting nutrient. In these forest soils nitrogen is mainly available as organic compounds which are not accessible to plants but to the ectomycorrhizal fungi. The fungal partners are able to break down proteins present in these soils by using extracellular proteinase. Thus, they can take up and assimilate organic nitrogen in the form of free amino acids and peptides which can then be transferred to the plant. Since ectomycorrhizal fungi strongly participate in nitrogen nutrition of the plant in these soils, it is necessary to understand uptake of organic nitrogen from the soil by the fungus (its subsequent transport to the plant) and its regulation. To identify the transporters involved in the uptake of organic nitrogen compounds by the fungal hyphae and their transfer to the plant, it was necessary to develop genomic tools. An oriented expression library was constructed from the mycelia of the ectomycorrhizal fungus Hebeloma cylindrosporum. In this work, the quality of this library was tested by DNA sequencing of ~500 ESTs and a sequence database was generated for the model fungus H. cylindrosporum (www.uni-tuebingen.de/plantphys/hebeloma/index.html). Furthermore the suitability of the library to identify Hebeloma genes via their function was demonstrated. Using Hebeloma cDNA libraries a gene encoding for an amino acid transporter (HcGAP1) was isolated and characterized. Two peptide transporters (HcPTR2A, -B) were also characterized. The characterization of these transporters by uptake experiments shows that they play a role in the import of organic nitrogen compounds into Hebeloma. Expression studies demonstrated that these transporters are regulated by different nitrogen sources. As extracellular proteinases play an important role in organic nitrogen availability, the proteinase activity of Hebeloma was characterized and a gene encoding for a putative extracellular proteinase was isolated. Finally, a method for Agrobacterium tumefaciens mediated transformation of Hebeloma was successfully established

Nitrogen transport in the ectomycorrhiza association: The Hebeloma cylindrosporum–Pinus pinaster model

International audienceThe function of the ectomycorrhizal mutualism depends on the ability of the fungal symbionts to take up nutrients (particularly nitrogen) available in inorganic and/or organic form in the soil and to translocate them (or their metabolites) to the symbiotic roots. A better understanding of the molecular mechanisms underlying nutrient exchanges between fungus and plant at the symbiotic interface is necessary to fully understand the function of the mycorrhizal symbioses. The present review reports the characterization of several genes putatively involved in nitrogen uptake and transfer in the Hebeloma cylindrosporum–Pinus pinaster ectomycorrhizal association. Study of this model system will further clarify the symbiotic nutrient exchange which plays a major role in plant nutrition as well as in resistance of plants against pathogens, heavy metals, drought stress, etc. Ultimately, ecological balance is maintained and/or improved with the help of symbiotic associations, and therefore, warrant further understanding

Fine mapping and chromosome walking towards the Ror1 locus in barley (Hordeum vulgare L.)

Recessively inherited loss-of-function alleles of the barley (Hordeum vulgare) Mildew resistance locus o (Mlo) gene confer durable broad-spectrum disease resistance against the obligate biotrophic fungal powdery mildew pathogen Blumeria graminis f.sp. hordei. Previous genetic analyses revealed two barley genes, Ror1 and Ror2, that are Required for mlo-specified resistance and basal defence. While Ror2 was cloned and shown to encode a t-SNARE protein (syntaxin), the molecular nature or Ror1 remained elusive. Ror1 was previously mapped to the centromeric region of the long arm of barley chromosome 1H. Here, we narrowed the barley Ror1 interval to 0.18 cM and initiated a chromosome walk using barley yeast artificial chromosome (YAC) clones, next-generation DNA sequencing and fluorescence in situ hybridization. Two non-overlapping YAC contigs containing Ror1 flanking genes were identified. Despite a high degree of synteny observed between barley and the sequenced genomes of the grasses rice (Oryza sativa), Brachypodium distachyon and Sorghum bicolor across the wider chromosomal area, the genes in the YAC contigs showed extensive interspecific rearrangements in orientation and order. Consequently, the position of a Ror1 homolog in these species could not be precisely predicted, nor was a barley gene co-segregating with Ror1 identified. These factors have prevented the molecular identification of the Ror1 gene for the time being.Johanna Acevedo-Garcia, Nicholas C. Collins, Nahal Ahmadinejad, Lu Ma, Andreas Houben, Pawel Bednarek, Mariam Benjdia, Andreas Freialdenhoven, Janine Altmüller, Peter Nürnberg, Richard Reinhardt, Paul Schulze‑Lefert, Ralph Panstrug

Characterization of a general amino acid permease from Hebeloma cylindrosporum

Besides a role in phosphate supply, ectomycorrhizas play a crucial role in nitrogen nutrition of plants. The ectomycorrhizal association between Hebeloma cylindrosporum and Pinus pinaster serves as a model system accessible to molecular manipulation. Hebeloma mycelium is able to take up and use amino acids as the sole nitrogen source. Suppression cloning allowed identification of a Hebeloma transporter (HcGAP1) mediating histidine uptake. HcGAP1 mediates secondary active uptake of a wide spectrum of different amino acids. The secondary active transport mechanism together with the expression in hyphae, but not in mycorrhizas, indicate a role in uptake of organic nitrogen from the soil

Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism

Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting thatmost effectors represent species-specific adaptations