CSEP0064/BEC1054 interacts with barley PR10 <i>in planta</i>.

A) Multiple amino acid sequence alignment of CSEP0064/BEC1054, CSEP0264/BEC1011 and CSEP0102. The alignment was generated by CLUSTALW using the mature polypeptides (i.e., the predicted N-terminal signal peptides removed). Amino acids that are identical between all three proteins are highlighted in black, amino acids shared by two of the three proteins are shown in gray. B) The host plant, barley, was used to perform a BiFC assay via particle bombardment-mediated transient gene expression in single leaf epidermal cells. As bait proteins, CSEP0064/BEC1054, CSEP0264/BEC1011 and CSEP0102 were used. The barley prey proteins were: Pathogenesis-Related protein 10 (PR10), eukaryotic Elongation Factor 1 Gamma (eEF1γ), eukaryotic Elongation Factor 1 Alpha (eEF1α) isoforms 1 and 3, malate dehydrogenase (MDH), Glutathione-S-Transferase (GST) and nucleoside diphosphate kinase (NDPK; negative control). BiFC signals were scored semiquantitatively by assigning transformed (DsRED-expressing) cells manually to one of four categories based on YFP fluorescence intensity. Center lines of box plots show the medians of the scores; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; outliers are represented by dots; crosses designate the arithmetic mean of the scores; and numbers of inspected cells are given below the boxes. C) Representative micrographs of BiFC-positive barley epidermal cells for the combinations CSEP0064/BEC1054-PR10 (upper panel), CSEP0064-PR10 (middle panel) and CSEP0064-eEF1α(3) (lower panel). For each combination a single focal plane and a maximum projection (z-stack) are shown. Scale bars are 20 μM.