Related Experiment Video

Updated: Feb 27, 2026

Identification of Post-translational Modifications of Plant Protein Complexes

Identification of Post-translational Modifications of Plant Protein Complexes

Published on: February 22, 2014

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

Xiaomin Hou¹, Yi Gao¹

¹Key Laboratory of Plant Biotechnology of Shandong Province, College of Life Sciences, Qingdao Agricultural University, Qingdao 266109. China.

Protein and Peptide Letters

|June 24, 2017

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 AtRabE1d13-185Q74L: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.

Keywords:

AtRabE1d13-185Q74L AvrPto Pseudomonas syringae Tomato bacterial speck disease pathogenic mechanism

More Related Videos

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Published on: May 24, 2018

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Published on: January 20, 2017

Related Concept Videos

Rab Proteins

Rab Proteins

Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...

Coat Assembly and GTPases

Coat Assembly and GTPases

Vesicles incorporate different coat protein subunits in different cell locations, which changes the properties of the coat, such as the shape and geometry of the transport vesicles. Thus, vesicle coat proteins also play a significant role in cargo selection.
Coat assembly depends on the local availability of phosphatidylinositol phosphates or PIPs and GTP-binding proteins. Adaptor proteins, which link the coat proteins to the membrane, bind to these PIPs and play a crucial role in controlling...

GPCRs Regulate Adenylyl Cylase Activity

GPCRs Regulate Adenylyl Cylase Activity

Some GPCRs transmit signals through adenylyl cyclase (AC), a transmembrane enzyme. AC helps synthesize second messenger cyclic adenosine monophosphate (cAMP). AC catalyzes cyclization reaction and converts ATP to cAMP by releasing a pyrophosphate. The pyrophosphate is further hydrolyzed to phosphate by the enzyme pyrophosphatase, which drives cAMP synthesis to completion. However, cAMP is rapidly degraded to 5′ AMP by the enzymes phosphodiesterase (PDE), preventing overstimulation of...

Small GTPases - Ras and Rho

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:

Rab Cascades

Rab Cascades

Rab GTPases act in a regulated cascade during membrane fusion, helping the lipid bilayers mix. The Rab family of proteins are active when bound to GTP, and inactive when bound to GDP. Hence, they act as guanine nucleotide-dependent molecular switches. Rab-GTP recognizes and binds to long or short-range tethering proteins to capture the target vesicle. These tethers coordinate with SNAREs on the vesicle and the target membrane to assemble the trans SNARE complex that locks the mixing bilayers.

Mechanism of Filopodia Formation

Mechanism of Filopodia Formation

Filopodia are thin, actin-rich cellular protrusions that play an important role in many fundamental cellular functions. They vary in their occurrence, length, and positioning in different cell types, suggesting their diverse roles.
Their main function is to guide migrating cells during normal tissue morphogenesis or cancer metastasis by recognizing and making initial contacts with the extracellular matrix. However, they can also act as stationary cell anchors or help to establish communication...

Articles linked to this work by shared authors, journal, and citation graph.

Nicotine Induces Pyroptosis in Mouse Primary Pulmonary Arterial Endothelial Cells through Cx43-Mediated Activation of the NLRP3/Caspase-1/GSDMD Signaling Pathway.

Chemical research in toxicology·2026

Association between body composition and metabolic dysfunction-associated steatotic liver disease in Chinese general population: a cross-sectional study stratified by sex and obesity status.

Frontiers in public health·2026

Interfacial microenvironment engineering of functionalized MOFs for immobilizing PET hydrolase ICCG to overcome the activity-stability trade-off in efficient PET depolymerization.

Bioresource technology·2026

Association between depression and anxiety and adverse obstetric and neonatal outcomes: A retrospective National Inpatient Sample database study.

Medicine·2026

The joint association of insulin resistance index and neutrophil percentage to albumin ratio with acute kidney injury after orthopedic surgery: a retrospective cohort study.

Frontiers in nutrition·2026

A clinical nomogram incorporating hyperhomocysteinemia for predicting severe delayed encephalopathy after acute carbon monoxide poisoning: a retrospective single-center study.

Frontiers in medicine·2026

See all related articles