Characterizing the nutrient exchange between symbionts is fundamental to understanding the lichen symbiosis. An important role is played by membrane-bound transport proteins, which regulate the flow of nutrients both within an organism and between symbionts. Because transporter proteins are easily identified bioinformatically and the functional assays required to characterize transport proteins are relatively straightforward, they are attractive targets for study. But the fact that lichens remain largely genetically intractable means that heterologous systems such as yeast or other model organisms must be used to confirm their function. The purpose of this work was to assemble a pipeline for the functional characterization of transporters important in the lichen symbiosis. Tripartite lichens were chosen because distinct suites of fungal transporters are required for the exchange of fixed carbon with the green algal partner and the exchange of fixed nitrogen with the cyanobacterial partner. Genes encoding transporters likely to be important for the exchange of carbon and nitrogen between symbionts were identified from the genomes and transcriptomes of Peltigera aphthosa and Peltigera britannica and annotated using information from the sequenced genomes of Coccomyxa and Nostoc. A subset of transporters with homology to sugar transporters, polyol transporters, ammonium transporters and amino acid transporters were amplified from the fungal, algal or bacterial symbionts of the lichens and were cloned and expressed in S. cerevisiae mutants for complementation assays. A further subset were expressed in Xenopus oocytes and tested via electrophysiology to determine functionality. This work will improve annotation of these transporter families in general and will help identify transporters important for this and other symbiotic interactions.
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