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. 2016 Jul 8;11(7):e0158856.
doi: 10.1371/journal.pone.0158856. eCollection 2016.

AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component With Homologs in Diverse Environmental Contexts

Free PMC article

AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component With Homologs in Diverse Environmental Contexts

Lindsay R Triplett et al. PLoS One. .
Free PMC article


Toxin-antitoxin (TA) systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Fig 1
Fig 1. AvrRxo1:Arc1 form a novel toxin-antitoxin system in the zeta:epsilon superfamily.
A) Alignment of the structures of AvrRxo1 (teal) and zeta toxin (magenta) demonstrate a conserved architecture. B) Alignment of ATP-binding domain shows complete conservation of functional residues, while C) only one of five residues predicted to coordinate the substrate are identical. D) Growth curves of E. coli BL21(DE3) co-transformed with pBAD33-avrRxo1 and either pDEST-arc1 or a pDEST control vector (pDESTcv) under conditions of avrRxo1 induction (arabinose) or repression (glucose). Data points and error bars represent the means and standard deviations of nine cultures. Growth experiments were performed three times on separate days with similar results.
Fig 2
Fig 2
Homologs of AvrRxo1 from multiple species suppress E. coli growth (A,B) and induce Rxo1-dependent cell death when transiently expressed in tobacco (C, D). A) Growth of BL21(DE3) carrying pDEST-AvrRxo1-Xoc, -Xe, -Ba, or -Xt, or pDESTcv, starting at 107 CFU/mL. B) Growth of the same strains starting at 106 CFU/mL. Error bars represent the standard deviation of 16 replicate cultures. Experiments were repeated three times on different days. C) N. benthamiana leaves four days after infiltration with Agrobacterium expressing YFP fusions of AvrRxo1-Xoc (1), -Ba(2), -Ac (3), and Xe (4), or YFP alone (5). D) N. benthamiana leaf in which the homologs from C were co-expressed with a YFP fusion of Rxo1, imaged two days after agroinfiltration.
Fig 3
Fig 3. Phylogeny of predicted AvrRxo1-like and Arc1-like proteins in plant pathogenic and environmental bacteria.
Neighbor-Joining tree of aligned amino acid sequences from 12 AvrRxo1 homologs (A) and 16 Arc1 homologs (B). Sequence accessions and strains are listed in Tables 1 and 3. The datasets consisted of 260 (A) and 74 (B) amino acids after gap elimination. Bootstrap percentages for 1000 replicates are shown next to branches. Units represent the number of amino acid substitutions per site.
Fig 4
Fig 4. An AvrRxo1 homolog from the myxobacterium Cystobacter fuscus causes watersoaking and Rxo1-mediated cell collapse after transient expression in N. benthamiana.
pEG104-AvrRxo1-Cf was expressed alone (A) and co-expressed with Rxo1 (B). 1 = AvrRxo1-Cf (OD600 = 0.1), 2 = AvrRxo1-Cf (OD600 = 0.4), 3 = GFP (OD600 = 0.4), 4 = AvrRxo1-Cf (OD600 = 0.1) and Rxo1 (OD600 = 0.4), 5 = GFP (OD600 = 0.1) and Rxo1 (OD600 = 0.4).

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Grant support

This project was supported by the Agriculture and Food Research Initiative Competitive grant no. 2014-67013-21564 of the USDA National Institute of Food and Agriculture ( to LRT and JEL and National Science Foundation ( grant no. IOS-0845283 to BZ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.