Subcellular localization and function of the Arabidopsis thaliana small GTPase RabE, a host interacting protein of the Pseudomonas syringae virulence effector AvrPto

Pseudomonas syringae pathovar tomato strain DC3000 (Pst DC3000) is a bacterial pathogen of tomato and of the model plant Arabidopsis thaliana. Like many Gram-negative bacterial pathogens of animals and plants, Pst DC3000 uses the conserved type III secretion system (TTSS) to deliver multiple virulence effector proteins directly into the host cell. Type III effectors collectively participate in causing disease, by mechanisms that are not well understood. Elucidating the virulence function of individual effectors is fundamental for understanding bacterial infection of plants.; Transgenic overexpression of AvrPto, one of Pst DC3000 virulence effector proteins, in Arabidopsis was previously shown to lead to suppression of basal defenses, thus enabling growth of non-pathogenic TTSS-defective bacteria in the transgenic plants. AvrPto interacts in the yeast two-hybrid system with the Arabidopsis RabE family of small GTPases, putative regulators of post-Golgi vesicle traffic to the plasma membrane. Although the function of RabE homologues in other eukaryotic organisms is well understood, the biological role of the Arabidopsis RabE proteins is obscure.; In this study, a live cell imaging approach was applied to investigate the subcellular localization of one of the five Arabidopsis RabE proteins, RabE1d, and of its mutant derivatives RabE1d-Q74L (predicted to be constitutively active) and RabE1d-S29N (predicted to be constitutively inactive), fused to the green fluorescent protein (GFP). Microscopic analysis and cell fractionation studies revealed that transgenically expressed GFP-RabE1d and endogenous RabE proteins are associated with the Golgi apparatus and the plasma membrane in Arabidopsis leaves. Strikingly, upon transgenic expression of AvrPto in planta, the Golgi-localized pool of GFP-RabE1d was greatly reduced and often undetectable. Furthermore, RabE1d overexpression could partially counteract the AvrPto-induced susceptibility to TTSS-defective bacteria. This work uncovered a novel association between AvrPto virulence function and subcellular distribution of the RabE protein.; To explore a possible role of RabE in plant growth, development and defense against Pst DC3000, transgenic Arabidopsis plants overexpressing RabE1d or its S29N and Q74L mutant variants were used. Overexpression of wild-type RabE1d or of RabE1d-S29N resulted in plants that were morphologically and developmentally indistinguishable from wild-type Arabidopsis and were not altered in disease resistance. Interestingly, Arabidopsis plants expressing the mutant RabE1d-Q74L gained a significant degree of resistance to Pst DC3000, while their growth and development were similar to those of wild-type plants. In contrast, RabE silencing drastically affected Arabidopsis leaf morphology and rosette size (suggesting a role for RabE in plant growth and development) and had a complex effect on host defense.; This study identified an original case of a virulence effector of a plant-pathogenic bacterium that alters subcellular localization of a putative regulator of intracellular trafficking. Additionally, functional study of RabE1d laid the basis for further characterization of the role of the entire RabE family of small GTPases in Arabidopsis.