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Summary• The expression of hydrophobin genes DGH1 , DGH2 and DGH3 in the lichenized fruitbody of Dictyonema glabratum (syn. Cora pavonia ) is reported here, as well as immunolocalization of the DGH1 protein.• In situ hybridization was conducted to determine expression patterns of hydrophobin genes DGH1 , DGH2 and DGH3 . The DGH1 protein was expressed in Escherichia coli and used to raise antibodies for immunolocalization of the DGH1 protein.• DGH1 and DGH2 are expressed by hyphae in the photobiont layer and the lower stratum. DGH3 is newly expressed by hyphae in the boundary layer. DGH1 is immunolocalized in the electron-dense outer layer of fungal walls lining gas-filled spaces in the photobiont layer, in aerial hyphae in the gas-filled lower stratum and in the transition zone between the two strata.• Dictyonema glabratum hydrophobins surround wall surfaces in the photobiont layer of the fruitbody. Hydrophobin layers probably function to keep interhyphal spaces water-free, seal the apoplast to enable water translocation, and define strata within the basidiocarp.

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