Figure 3. Identity of the C-terminus is critical for MLO functionality.

(A) Amino acid sequence alignment of the C-termini of barley (MLO), wheat (TaMLO-A1 and TaMLO-B1), rice (OsMLO1) and Arabidopsis (AtMLO11) MLO isoforms. The previously characterized CaMBD is indicated by a bar above the sequence. Two MLO serine residues (Ser⁴¹⁷ and Ser⁴⁵³) identified to be crucial for full MLO function (see Figure 2D) are highlighted by an asterisk above the sequence. (B) Functional assay of MLO variants. Leaf segments of the powdery-mildew-resistant barley cultivar BC Ingrid mlo-5 were bombarded with the bifunctional plasmid pUGLUM (encoding the GFP reporter plus wild-type MLO) or a mutant version thereof (encoding GFP plus TaMLO-A1 CT, TaMLOB1 CT, OsMLO1 CT or AtMLO11 CT). Leaves were then inoculated with Bgh A6 and GFP-fluorescent cells were inspected for fungal structures as described in the Materials and methods section. (C and E) Assessment of MLO protein accumulation. Relative accumulation of wild-type MLO, mutant version MLO-1, as well as chimaeras TaMLO-A1 CT, TaMLO-B1 CT, OsMLO1 CT, AtMLO11 CT, CT swap, CT swap A417S and CT swap A453S was determined by dual-luciferase assays of transfected A. thaliana protoplasts as described in the Materials and methods section. Note that the values obtained for MLO and MLO-1 result from a common experiment with the constructs shown in Figure 4(C). (D) Functional assay of single-amino-acid-substitution MLO variants. Leaf segments of the powdery-mildew-resistant barley cultivar BC Ingrid mlo-5 were co-bombarded with a GUS reporter construct and the bifunctional plasmid pUGLUM (encoding the GFP reporter plus wild-type MLO) or mutant versions thereof (encoding variants CT swap, CT swap A417S or CT swap A453S). Leaves were then inoculated with Bgh K1 and were stained for GUS activity and fungal structures as described in the Materials and methods section. (F) Schematic representation of the domain-swap constructs TaMLO-A1 CT, TaMLOB1 CT, OsMLO1 CT, AtMLO11 CT, CT swap, CT swap A417S and CT swap A453S. Black bends illustrate barley MLO portions, whereas light-grey bends depict the respective heterologous fraction. Black dots symbolize single-amino-acid-replacements A417S or A453S.