Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein

Abstract

We performed a structure-function analysis of the plasma membrane-localized plant-specific barley (Hordeum vulgare) MLO (powdery-mildew-resistance gene o) protein. Invariant cysteine and proline residues, located either in extracellular loops or transmembrane domains that have been conserved in MLO proteins for more than 400 million years, were found to be essential for MLO functionality and/or stability. Similarly to many metazoan G-protein-coupled receptors known to function as homo- and hetero-oligomers, FRET (fluorescence resonance energy transfer) analysis revealed evidence for in planta MLO dimerization/oligomerization. Domain-swap experiments with closely related wheat and rice as well as diverged Arabidopsis MLO isoforms demonstrated that the identity of the C-terminal cytoplasmic tail contributes to MLO activity. Likewise, analysis of a progressive deletion series revealed that integrity of the C-terminus determines both MLO accumulation and functionality. A series of domain swaps of cytoplasmic loops with the wheat (Triticum aestivum) orthologue, TaMLO-B1, provided strong evidence for co-operative loop-loop interplay either within the protein or between MLO molecules. Our data indicate extensive intramolecular co-evolution of cytoplasmic domains in the evolutionary history of the MLO protein family.

Figure 1. Invariant cysteine and proline residues are essential for MLO function and/or protein accumulation

(A) Schematic representation of MLO depicting strictly conserved residues within the MLO protein family. The light-grey box represents the lipid bilayer, whereas smaller dark-grey boxes symbolize the seven-transmembrane domains. Invariant amino acids of a sample of 38 full-size MLO sequences (see text) are shown as circles labelled using the one-letter amino acid code. Numbering of amino acids corresponds to barley MLO [4]. (B) Functional assay of single-amino-acid-substitution 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 either the double-mutant MLO E104N/R106S or one of the four cysteine→alanine mutants). 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 and mutant versions MLO-1, C86A, C98A, C114A, C367A, P287G and P395G was determined by dual-luciferase assays of transfected Arabidopsis thaliana protoplasts as described in the Materials and methods section. (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 a mutant version thereof (encoding P287G or P395G single-amino-acid-substitution variants). Leaves were then inoculated with Bgh K1 and stained for GUS activity and fungal structures as described in the Materials and methods section.

Figure 2. FRET analysis reveals MLO homo-oligomerization

(A) Quantitative FRET APB analysis. Background FRET (white bars) and FRET efficiencies (black bars) were calculated from barley leaf epidermal cells co-expressing the constructs shown below the graph (B). Results are means±S.D. for 26–80 cells each. (B) Schematic representation of constructs used for FRET analysis. MLO is depicted by its serpentine structure, CaM as an oval, and YFP and CFP fluorophores as light-grey (upper panel) and dark-grey (lower panel) ribbon models respectively. Two dots in the C-terminal tail of MLO symbolize the two amino acid substitutions in the L420R/W423R MLO CaMBD mutant variant.

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.

Figure 4. Integrity of the C-terminus is critical for MLO functionality

(A) Schematic representation of the MLO C-terminus and indication of the derived C-terminal truncation variants. The light-grey box symbolizes the lipid bilayer. Individual amino acids are shown as circles labelled using the one-letter amino acid code. The last amino acid of the respective indicated constructs carrying C-terminal truncations is highlighted in dark grey. (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 either RDM (Δ40), VHL (Δ67), SPM (Δ77), QMI (Δ89), KVR (Δ99), NWR (Δ109) or DEQ (Δ118)]. 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) Assessment of MLO protein accumulation. Relative accumulation of wild-type MLO, mutant version MLO-1, as well as variants RDM (Δ40), VHL (Δ67), SPM (Δ77), QMI (Δ89), KVR (Δ99), NWR (Δ109) and DEQ (Δ118) carrying increasing C-terminal truncations was determined by dual-luciferase assays of transfected A. thaliana protoplasts as described in the Materials and methods section.

Figure 5. Domain-swap analysis with intracellular domains of barley MLO and TaMLO-B

(A) Amino acid sequence alignment of the first to third intracellular loop (IC1–IC3), as well as the CT of MLO and TaMLO-B1. (B) 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. (C) Assessment of MLO protein accumulation. Relative accumulation of wild-type MLO and mutant version MLO-1, as well as variants TaMLO-B1, MLO IC1, MLO IC2, MLO CT, MLO IC1+IC2, MLO IC1+CT, MLO IC2+CT and MLO IC1+IC2+CT 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 Figures 2(C) and 2(E). (D) Schematic representation of the domain-swap constructs MLO IC1, MLO IC2, MLO CT, MLO IC1+IC2, MLO IC1+CT, MLO IC2+CT and MLO IC1+IC2+CT. Black bends illustrate MLO portions, whereas light-grey bends depict the respective TaMLO-B1 fraction.