Investigation on the Interaction of Pseudomonas syringae Effector AvrPto with AtRabE1d GTPase.

ABSTRACT

BACKGROUND: The tomato bacterial speck is a worldwide disease. It is caused by the infection of pathogenic Pseudomonas syringae pv. tomato which delivers the effector AvrPto into the host cells via the type III secretion system. AvrPto interacts with a Rab8 subfamily protein in the GTP-bound form and participates in the response to pathogen infection, but the pathogenic mechanism involved remains elusive. OBJECTIVES: The main objective of this study was to investigate on the interrelationship of AvrPto with AtRabE1d and Pto, which would allow us to have a deeper understanding of the pathogen mechanism of bacterial speck mediated by avirulent AvrPto and provides theoretic support for the prevention and cure of tomato bacterial speck disease. METHODS: AvrPto8-159 and AtRabE1d13-185Q74L proteins were expressed in Escherichia coli expression system, purified via nickel affinity chromatography and size exclusion chromatography, and identified by SDS-PAGE. The interaction of AvrPto8-159 with AtRabE1d13-185Q74L was confirmed in vitro based on the fluorescence resonance energy transfer. In addition, the affinity of AvrPto8-159 with AtRabE1d13-185Q74LGTP was determined using the fluorescence polarization based equilibrium titration. The comparison of the complex structural model of AtRabE1d with AvrPto, which was docked and refined by Patchdock and FireDock softwares. RESULTS: AvrPto8-159 and AtRabE1d13-185Q74L can be expressed and purified well in E.coli. FRET between AvrPto8-159 with GFP-tag and mantGTP-load AtRabE1d13-185Q74L was observed slightly in vitro for the first time. On the other hand, deploying DsRed of AtRabE1d13-185Q74L as FRET partner with GFP, AvrPto was shown to interact with AtRabE1d more significantly with increasing concentrations of DsRed-AtRabE1d13-185Q74L. The equilibrium dissociation constant of AvrPto8-159 with AtRabE1d13-185Q74LGTP was determined to be 13.5 µM. CONCLUSION: This work reports preparation and interaction of AvrPto8-159 with AtRabE1d13- 185Q74L. AvrPto8-159 exhibited medium affinity with AtRabE1d13-185Q74L based on the fluorescence polarization. From the structural model of the complex AvrPtoAtRabE1d, AvrPto interacted with AtRabE1d and Pto proteins with completely different biding sites.