Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein.
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.
Elliott
Candace
C
The Sainsbury Laboratory, John Innes Centre, Colney, Norwich, NR4 7UH, UK.
Müller
Judith
J
Miklis
Marco
M
Bhat
Riyaz A
RA
Schulze-Lefert
Paul
P
Panstruga
Ralph
R
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D001467:Hordeum / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
C104922:MLO protein, Hordeum vulgare; D010940:Plant Proteins; D011392:Proline; D003545:Cysteine