Supporting Information

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Fig. 5. (A–F). Accumulation pattern of barley plasma membrane proteins upon Bgh challenge. Barley epidermal cells expressing Aquaporin 1-YFP (A), Aquaporin 2-YFP (B), receptor like kinase-YFP (C), lipid transfer protein-YFP (D), cytochrome b561-YFP (E), or SYP132-YFP (F) were challenged with Bgh conidiospores. At 10-14 hpi, epiphytic fungal structures were stained with propidium iodide (red). The stage of fungal development on a cross-sectioned epidermal cell is depicted schematically above the micrographs. The barley cDNAs were selected due to presence of respective EST clones in the barley HO cDNA library (Institute of Crop Genetics and Crop Plant Research, Gatersleben, Germany) that was generated from pathogen-challenged leaf epidermal peels, thus ensuring that all genes used are expressed in the barley leaf epidermis. Full-sized cDNA clones derived from of either this or other barley cDNA libraries were used for generation of the YFP fusion constructs. Plasma membrane localization is predicted for all tested proteins (aquaporins, barley cDNAs HG01L04 and HE01D16, GenBank accession nos. BQ463875 and BQ461093 (1); Xa21-like receptor kinase, barley cDNA HO04G20, GenBank accession no. CD053483 (2); lipid transfer protein, barley cDNA HO14B02, GenBank accession no. CD057067 (3); cytochrome b561, barley cDNA HM05C15, GenBank accession no. BU993960 (4); and syntaxin SYP132, barley cDNA HO12N23, GenBank accession no. CD056658 (5).

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Fig. 6. Variation in FRET-APB efficiency between MLO-YFP and CFP-CaM at different sample sites. FRET-APB efficiencies were determined at various sites in barley epidermal cells coexpressing MLO-YFP and CFP-CaM. FRET efficiencies (red bars) and background FRET (blue bars) are shown. The 80 sample sites result from at least 50 transformed leaf epidermal cells.

Fig. 7. MLO-YFP/CFP-CaM FRET efficiency varies between subcellular regions. Barley epidermal cells coexpressing MLO-YFP and CFP-CaM were challenged with Bgh conidiospores. FRET was quantified using APB in various subcellular regions as schematically depicted above the graph at 14-24 hpi. Mean FRET efficiencies (black bars) and background FRET (white bars) ± SD from 50 to 100 sample sites are shown. Also shown is the FRET efficiency between cytoplasmic CFP and YFP determined under the same conditions. MLO is depicted by its serpentine structure, CaM is a purple circle, and CFP and YFP fluorophores are cyan and yellow ribbon models, respectively. 1, area around the attempted fungal penetration site; 2, area at a distance from attempted fungal penetration sites; EM, endomembranes; N, area around the nucleus; V, moving vesicular structures; s, spore.