Comprehensive comparative assessment of theArabidopsis thalianaMLO2-calmodulin interaction by variousin vitroandin vivoprotein-protein interaction assays

AbstractMildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the fungal powdery mildew pathogen. In many angiosperm plant species, loss-of-functionmlomutants confer durable broad-spectrum resistance against the powdery mildew disease. Barley Mlo is known to interactviaa cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calcium-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction betweenArabidopsis thalianaMLO2 and CAM2 using seven different types ofin vitroandin vivoprotein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2CT), harboring the CAMBD, or the MLO2 full-length protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CTLW/RR mutant variants in comparison to the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system andin plantabimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein-protein interaction assays with wild-type and mutant versions of an integral membrane protein.