Detection and Functional Characterization of a 215 Amino Acid N-Terminal Extension in the Xanthomonas Type III Effector XopD

Joanne Cannone,Laurent D Noel ,Carole Pichereaux,Susana Rivas,Daniel Marino,Dominique Roby,Michel Rossignol,Ignacio Arechaga

doi:10.1371/journal.pone.0015773

Abstract

During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD1-760) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD216-760). Furthermore, the N-terminal extension of XopD, which is absent in XopD216-760, is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta.

Highlights

Plants have developed a complex defence network to fight off invading pathogens
We demonstrate that the functional protein expressed by Xanthomonas campestris pathovar vesicatoria (Xcv) presents a previously overlooked N-terminal extension of 215 amino acids that is essential for its T3SS-dependent secretion and virulence-associated functions in the plant cell
Close inspection of the Xcv 85-10 genomic sequence neighbouring the annotated xopD gene shows that upstream of the annotated starting codon AUG, there are 6 AUG, 1 GUG and 5 UUG additional codons that are in frame and may potentially be used to start translation of the protein (Figure 1C)

Summary

Introduction

The first layer of plant defence involves recognition of PAMPs (Pathogen-Associated Molecular Patterns), defined as invariant epitopes within molecules that are fundamental to pathogen fitness and widely distributed among different microbes [1]. This recognition, previously known as basal defence, is referred to as PAMP-triggered immunity (PTI) [2]. Plants acquired the ability to recognize directly or indirectly effectors through resistance (R) proteins This recognition response is associated with the long-standing gene-forgene hypothesis, and more recently with the guard hypothesis [10], and is known as effector-triggered immunity (ETI). Several T3Es from Gram-negative phytopathogenic bacteria have been shown to suppress ETI [11], suggesting that T3Es may have multiple targets or that they target shared components between PTI and ETI

Methods

Results

Conclusion