The association between Dioscorea sansibarensis and Orrella dioscoreae as a model for hereditary leaf symbiosis.

Hereditary, or vertically-transmitted, symbioses affect a large number of animal species and some plants. The precise mechanisms underlying transmission of functions of these associations are often difficult to describe, due to the difficulty in separating the symbiotic partners. This is especially the case for plant-bacteria hereditary symbioses, which lack experimentally tractable model systems. Here, we demonstrate the potential of the leaf symbiosis between the wild yam Dioscorea sansibarensis and the bacterium Orrella dioscoreae (O. dioscoreae) as a model system for hereditary symbiosis. O. dioscoreae is easy to grow and genetically manipulate, which is unusual for hereditary symbionts. These properties allowed us to design an effective antimicrobial treatment to rid plants of bacteria and generate whole aposymbiotic plants, which can later be re-inoculated with bacterial cultures. Aposymbiotic plants did not differ morphologically from symbiotic plants and the leaf forerunner tip containing the symbiotic glands formed normally even in the absence of bacteria, but microscopic differences between symbiotic and aposymbiotic glands highlight the influence of bacteria on the development of trichomes and secretion of mucilage. This is to our knowledge the first leaf symbiosis where both host and symbiont can be grown separately and where the symbiont can be genetically altered and reintroduced to the host.

Agrobacterium tumefaciens-mediated transformation of Nigrospora sp. isolated from switchgrass leaves and antagonistic toward plant pathogens.

Nigrospora is a diverse genus of fungi colonizing plants through endophytic, pathogenic, or saprobic interactions. Endophytic isolates can improve growth and development of host plants, as well as their resistance to microbial pathogens, but exactly how they do so remains poorly understood. Developing a reliable transformation method is crucial to investigate these mechanisms, in particular to identify pivotal genes for specific functions that correlate with specific traits. In this study, we identified eight isolates of Nigrospora sp. internally colonizing the leaves of switchgrass plants cultivated in North Carolina. Using an Agrobacterium tumefaciens-mediated transformation approach with control and GFP-expressing vectors, we report the first successful transformation of two Nigrospora isolates. Finally, we demonstrate that wild-type and transgenic isolates both negatively impact the growth of two plant pathogens in co-culture conditions, Bipolaris maydis and Parastagonospora nodorum, responsible for the Southern Leaf Blight and Septoria Nodorum Blotch diseases, respectively. The GFP-transformed strains developed here can therefore serve as accurate reporters of spatial interactions in future studies of Nigrospora and pathogens in the plant. Finally, the transformation method we describe lays the foundation for further genetic research on the Nigrospora genus to expand our mechanistic understanding of plant-endophyte interactions.

Secrets of the fungus-specific potassium channel TOK family.

Several families of potassium (K+) channels are found in membranes of all eukaryotes, underlining the importance of K+ uptake and redistribution within and between cells and organs. Among them, TOK (tandem-pore outward-rectifying K+) channels consist of eight transmembrane domains and two pore domains per subunit organized in dimers. These channels were originally studied in yeast, but recent identifications and characterizations in filamentous fungi shed new light on this fungus-specific K+ channel family. Although their actual function in vivo is often puzzling, recent works indicate a role in cellular K+ homeostasis and even suggest a role in plant-fungus symbioses. This review aims at synthesizing the current knowledge on fungal TOK channels and discussing their potential role in yeasts and filamentous fungi.

New insights into HcPTR2A and HcPTR2B, two high-affinity peptide transporters from the ectomycorrhizal model fungus Hebeloma cylindrosporum.

While plants mainly rely on the use of inorganic nitrogen sources like ammonium and nitrate, soil-borne microorganisms like the ectomycorrhizal fungus Hebeloma cylindrosporum can also take up soil organic N in the form of amino acids and peptides that they use as nitrogen and carbon sources. Following the previous identification and functional expression in yeast of two PTR-like peptide transporters, the present study details the functions and substrates of HcPTR2A and HcPTR2B by analysing their transport kinetics in Xenopus laevis oocytes. While both transporters mediated high-affinity di- and tripeptide transport, HcPTR2A also showed low-affinity transport of several amino acids-mostly hydrophobic ones with large side chains.

Plant potassium nutrition in ectomycorrhizal symbiosis: properties and roles of the three fungal TOK potassium channels in Hebeloma cylindrosporum.

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K+ ) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K+ nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K+ uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem-pore outward-rectifying K+ (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two-electrode voltage-clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K+ in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K+ from the fungus towards the plant.