Figure 3

Biological activity of Mlo variants with single amino acid substitutions in the second and third cytoplasmic loop. A. Pairwise amino acid alignment of the third cytoplasmic loop of barley Mlo and AtMLO2. mlo-27 (G318E), mlo-29 (P334L) and mlo-33 (A306T) denote previously known barley mlo mutant sites. Highlighted in black are identical amino acid residues, in grey residues with similar biochemical properties. Lines indicate amino acids selected for site-directed mutagenesis. B. Relative protein accumulation of Mlo protein variants. Protein accumulation was determined using the dual luciferase assay using wild type Mlo as a positive control (set as 100%) and the Mlo-1 mutant variant (characterized by the W162R amino acid substitution) as a negative control. Values represent mean ± standard deviation based on 4 independent experiments with 2 technical replicates each. Asterisks indicate a statistically significant difference (p < 0.05) from Mlo wild type according to Student's t-test. C. Functional assay of Mlo protein variants. Leaf segments of the powdery mildew resistant barley line BCI mlo-3 were co-bombarded with the pUbi-GUS reporter plasmid and a plasmid encoding the indicated Mlo protein variant. Host cell entry was scored in GUS-stained cells attacked by powdery mildew sporelings. Expression of wild type Mlo served as a positive control, expression of GUS alone as a negative control. Values represent mean ± standard deviation based on at least 3 experiments with each ca. 100 investigated GUS-positive cells/construct. Asterisks indicate a statistically significant difference (p < 0.05) from the GUS control according to Student's t-test.