Expression, Purification and in vitro Enzyme Activity Assay of Plant Derived GTPase

Abstract

Based on gene expression data after biotic stress, the GTPase RabA4c has been suggested to regulate pathogen-induced callose biosynthesis in the model organism Arabidopsis thaliana. We studied the function of RabA4c in its native and dominant negative (dn) isoform. In planta, RabA4c overexpression prevented penetration of the virulent powdery mildew Golovinomyces cichoracearum into epidermal leaf cells. This penetration resistance was caused by enhanced callose deposition at sites of attempted fungal penetration at early time points of infection. By contrast, RabA4c (dn) overexpression did not increase callose deposition or penetration resistance. In this protocol, we describe the expression, purification and activity assay of the heterologously expressed GTPase RabA4c from A. thaliana based on the publication Ellinger et al. (2014). We fused RabA4c to the fluorophore mCitrine and expressed this protein in the yeast strain Pichia pastoris GS115. For purification of RabA4c, we used the GFP-Trap_A kit (Chromo Tek) which specifically binds to GFP derivatives like mCitrine. The enzyme activity assay was done by using the GTPase Assay Kit from Innova Biosciences. In general, we followed the instructions made by the manufacturers.