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 AtRabE1d13-185Q74L:GTP 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 AtRabE1d13-185Q74L:GTP 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 AvrPto:AtRabE1d, AvrPto interacted
with AtRabE1d and Pto proteins with completely different biding sites.