The Type III Effector XopAJ from //Xanthomonas//

Authors: Ralf Koebnik & Trainees from the 2^nd EuroXanth Training School (Daiva Burokienė, Edyta Đermić, Dagmar Stehlikova, Mariya Stoyanova)
Internal reviewer: Joël F. Pothier
Expert reviewer: Lindsay Triplett

Class: XopAJ
Family: XopAJ
Prototype: AvrRxo1 (Xanthomonas oryzae pv. oryzicola; strain BLS256)
GenBank ID: AAQ97593.1 (421 aa)
RefSeq ID: WP_014504815.1 (421 aa)
Synonym: AvrRxo1
3D structure: 4Z8Q, 4Z8T, 4Z8U, 4Z8V (Han et al., 2015)

Maize lines that contain the single dominant gene Rxo1 exhibit a rapid hypersensitive response (HR) after infiltration with the nonhost rice bacterial streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc) and some strains of the maize pathogen Paraburkholderia andropogonis, but not with the rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) (Zhao et al., 2004). The avirulence effector gene that corresponds to Rxo1, designated avrRxo1, was identified in an Xoc genomic library (Zhao et al., 2004).

When expressed in an Xoo hrpC mutant that is deficient in the type III secretion system, avrRxo1 did not elicit the HR, indicating that the avrRxo1-Rxo1 interaction is dependent on type III secretion (Zhao et al., 2004). Transient expression in maize lines carrying Rxo1 resulted in cell death, suggesting that AvrRxo1 functions from inside maize cells to elicit Rxo1-dependent pathogen recognition (Zhao et al., 2004).

No data available.

• When introduced into Xoo, clones containing avrRxo1 induced an HR on maize with Rxo1, but not on maize without Rxo1 (Zhao et al., 2004).
• Rxo1 has a nucleotide-binding site-leucine-rich repeat structure, similar to many previously identified R genes (Zhao et al., 2005). Rxo1 functions after transfer as a transgene to rice, demonstrating the feasibility of nonhost R gene transfer between cereals (Zhao et al., 2005; Xie et al., 2007).
• AvrRxo1 is cytotoxic when expressed in yeast and caused chlorosis and patches of cell death in the infiltrated leaf areas upon transient expression in tomato and Nicotiana benthamiana (Salomon et al., 2011).
• Variants of AvrRxo1 were found to suppress the HR caused by the non-host resistance recognition of Xoo by N. benthamiana (Liu et al., 2014).
• Among four avrRxo1 alleles from different Xoc strains, toxicity is abolished by a single amino acid substitution at residue 344 in two AvrRxo1 variants (Liu et al., 2014).
• The ATP/GTP binding site motif A and the NLS are required for both the avirulence activity and the suppression of non-host resistance (Liu et al., 2014).
• AvrRxo1 has a T4 polynucleotide kinase domain and a structure homologous to that of Zeta toxins, and expression of AvrRxo1 suppresses bacterial growth in a manner dependent on the kinase motif (Han et al., 2015).
• The gene product of the adjacent gene, AvrRxo1-ORF2 aka Arc1, suppresses the bacteriostatic activity of AvrRxo1 in bacterial cells (Han et al., 2015).
• AvrRxo1 and its binding partner Arc1 function as a toxin-antitoxin system when expressed in Escherichia coli (Triplett et al., 2016). Bacterial toxicity is dependent on bacterial growth rate and conferred by diverse AvrRxo1 homologs from X. euvesicatoria, B. andropogonis, and X. translucens.
• XopAJ_Xcv85-10 inhibited activation of a PTI-inducible promoter by the bacterial peptide elf18 in Arabidopsis protoplasts and by flg22 in tomato protoplasts. This effector inhibited flg22-induced callose deposition in planta and enhanced disease symptoms caused by attenuated Pseudomonas syringae bacteria (Popov et al., 2016).
• AvrRxo1 is a kinase that converts NAD to 3'-NADP and NAADP to 3'-NAADP. Mutation of the catalytic aspartic acid residue D₁₉₃ abolished AvrRxo1 kinase activity and several phenotypes of AvrRxo1, including toxicity in yeast, bacteria, and plants, suppression of the flg22-triggered ROS burst, and ability to trigger an R gene-mediated hypersensitive response in rice. A mutation in the Walker A ATP-binding motif, which reduced 3'-NADP production by roughly 90%, abolished the toxicity of AvrRxo1 in bacteria, yeast, and plants. However, this mutation did not abolish the virulence enhancement, ROS suppression, or HR-triggering phenotypes of AvrRxo1. These results demonstrate that AvrRxo1 kinase activity is required for all the known phenotypes of AvrRxo1, but that toxicity is dose-dependent (Shidore et al., 2017).
• AvrRxo1 targets the cysteine protease RD21A, which is required for drought-induced immunity (Liu et al., 2020).
• AvrRxo1 enhances Xoc virulence and inhibits stomatal immunity by targeting and degrading rice OsPDX1 (pyridoxal phosphate synthase), thereby reducing vitamin B6 (VB6) levels in rice (Liu et al., 2022).

Transient expression of avrRxo1 in onion cells after biolistic delivery revealed that the protein product was associated with the plasma membrane (Zhao et al., 2004). However, later studies using fluorescently-tagged AvrRxo1 indicate localization in the nucleus and cytoplasm as well (Liu et al., 2014, Triplett et al., 2016, Liu et al., 2020).

AvrRxo1 has a T4 polynucleotide kinase domain (Han et al., 2015; Wu et al., 2015). AvrRxo1 is an ATP-dependent protease (Liu et al., 2022).

AvrRxo1 is a phosphotransferase that produces two novel metabolites by phosphorylating nicotinamide/nicotinic acid adenine dinucleotide at the adenosine 3'-hydroxyl group. Both products of AvrRxo1, 3'-NADP and 3'-nicotinic acid adenine dinucleotide phosphate (3'-NAADP), had been used before as inhibitors or signaling molecules but were regarded as “artificial” compounds until then (Schuebel et al., 2016). AvrRxo1 has weak phosphorylation activity on some other nucleotides including ATP (Scheubel et al. 2016)

AvrRxo1 phosphorylates NAD in planta, and its kinase catalytic sites are necessary for toxicity, suppression of PAMP-triggered immunity, and activation of Rxo1-mediated resistance (Shidore et al., 2017). In a metabolomic profile, 3'-NADP accumulated upon expression of AvrRxo1 in E. coli, yeast, N. benthamiana and in rice leaves infected with avrRxo1-expressing strains of X. oryzae, suggesting that the AvrRxo1 product is not utilized or degraded by the cell (Shidore et al., 2017). 3'-NADP was the only metabolite observed to accumulate in an avrRxo1-dependent manner, and it is not known whether NAADP is phosphorylated by AvrRxo1 in planta (Shidore et al., 2017).

Molecular modeling was used to decipher structural mechanisms of AvrRxo1-Rxo1 interaction (Bahadur & Basak, 2014).

The gene product of the adjacent gene, AvrRxo1-ORF2 aka Arc1, binds AvrRxo1, but binding is structurally different from typical effector-binding chaperones, in that it has a distinct fold containing a novel kinase-binding domain (Han et al., 2015).

AvrRxo1 interacts with the Arabidopsis thaliana ubiquitin E3 ligase SINAT4 and the cysteine protease RD21A during transient expression in N. benthamiana. Interaction enhanced SINAT4 activity and promoted the degradation of RD21A in vivo, in a manner dependent on the AvrRxo1 ATP-binding motif (Liu et al., 2020).

AvrRxo1 interacts with OsPDX1.2 in a yeast two-hybrid assay and in planta, as assessed by split YFP and coIP assays (Liu et al., 2022).

Yes (e.g. X. alfalfae, X. axonopodis, X. bromi, X. euvesicatoria, X. oryzae, X. translucens).

AvrRxo1 appears to be widely conserved in Asian strains of Xoc but much less present in African strains, which implies that deployment of Rxo1-containing varieties may not be an appropriate breeding strategy for controlling bacterial leaf streak disease in Africa (Wonni et al., 2014).

AvrRxo1 is conserved in nearly all strains of X. euvesicatoria, but is incompletely distributed in other species surveyed (Triplett et al. 2016, Barak et al. 2016). Strains with inactivated avrRxo1 genes were frequently observed to harbor sequence insertions in the toxic avrRxo1 gene, while the toxin-protective arc1 gene remained intact (Triplett et al., 2016).

Yes (Acidovorax spp., Paraburkholderia andropogonis) (Triplett et al., 2016).

Homologs of the avrRxo1:arc1 operon in which the avrRxo1 homolog lacks a type III secretion signal are found in other environmental microbes, including the filamentous myxobacteria Cystobacter fuscus and uncultured candidate Saccharimonas and Parcubacteria spp. (Triplett et al. 2016).

Yes (e.g. X. alfalfae, X. axonopodis, X. bromi, X. euvesicatoria, X. oryzae, X. translucens).

AvrRxo1 appears to be widely conserved in Asian strains of Xoc but much less present in African strains, which implies that deployment of Rxo1-containing varieties may not be an appropriate breeding strategy for controlling bacterial leaf streak disease in Africa (Wonni et al., 2014).

AvrRxo1 is conserved in nearly all strains of X. euvesicatoria, but is incompletely distributed in other species surveyed (Triplett et al. 2016, Barak et al. 2016). Strains with inactivated avrRxo1 genes were frequently observed to harbor sequence insertions in the toxic avrRxo1 gene, while the toxin-protective arc1 gene remained intact (Triplett et al., 2016).

Yes (Acidovorax spp., Paraburkholderia andropogonis) (Triplett et al., 2016).

Homologs of the avrRxo1:arc1 operon in which the avrRxo1 homolog lacks a type III secretion signal are found in other environmental microbes, including the filamentous myxobacteria Cystobacter fuscus and uncultured candidate Saccharimonas and Parcubacteria spp. (Triplett et al. 2016).

Bahadur RP, Basak J (2014). Molecular modeling of protein-protein interaction to decipher the structural mechanism of nonhost resistance in rice. J. Biomol. Struct. Dyn. 32: 669-681. DOI: 10.1080/07391102.2013.787370

Han Q, Zhou C, Wu S, Liu Y, Triplett L, Miao J, Tokuhisa J, Deblais L, Robinson H, Leach JE, Li J, Zhao B (2015). Crystal structure of Xanthomonas AvrRxo1-ORF1, a type III effector with a polynucleotide kinase domain, and its interactor AvrRxo1-ORF2. Structure 23: 1900-1909. DOI: 10.1016/j.str.2015.06.030

Liu H, Chang Q, Feng W, Zhang B, Wu T, Li N, Yao F, Ding X, Chu Z (2014). Domain dissection of AvrRxo1 for suppressor, avirulence and cytotoxicity functions. PLoS One 9: e113875. DOI: 10.1371/journal.pone.0113875

Liu H, Lu C, Li Y, Wu T, Zhang B, Liu B, Feng W, Xu Q, Dong H, He S, Chu Z, Ding X (2022). The bacterial effector AvrRxo1 inhibits vitamin B6 biosynthesis to promote infection in rice. Plant Commun. 3: 100324. DOI: 10.1016/j.xplc.2022.100324

Liu Y, Wang K, Cheng Q, Kong D, Zhang X, Wang Z, Wang Q, Qi X, Yan J, Chu J, Ling H, Li Q, Miao J, Zhao B (2020). Cysteine protease RD21A regulated by E3 ligase SINAT4 is required for drought-induced resistance to Pseudomonas syringae in Arabidopsis. J. Exp. Bot. 71: 5562-5576. DOI: 10.1093/jxb/eraa255

Popov G, Fraiture M, Brunner F, Sessa G (2016). Multiple Xanthomonas euvesicatoria type III effectors inhibit flg22-triggered immunity. Mol. Plant Microbe Interact. 29: 651-660. DOI: 10.1094/MPMI-07-16-0137-R

Salomon D, Dar D, Sreeramulu S, Sessa G (2011). Expression of Xanthomonascampestris pv. vesicatoria type III effectors in yeast affects cell growth and viability. Mol. Plant Microbe Interact. 24: 305-314. DOI: 10.1094/MPMI-09-10-0196

Schuebel F, Rocker A, Edelmann D, Schessner J, Brieke C, Meinhart A (2016). 3'-NADP and 3'-NAADP, two metabolites formed by the bacterial type III effector AvrRxo1. J. Biol. Chem. 291: 22868-22880. DOI: 10.1074/jbc.M116.751297

Shidore T, Broeckling CD, Kirkwood JS, Long JJ, Miao J, Zhao B, Leach JE, Triplett LR (2017). The effector AvrRxo1 phosphorylates NAD in planta. PLoS Pathog. 13: e1006442. DOI: 10.1371/journal.ppat.1006442

Triplett LR, Shidore T, Long J, Miao J, Wu S, Han Q, Zhou C, Ishihara H, Li J, Zhao B, Leach JE (2016). AvrRxo1 Is a bifunctional type III secreted effector and toxin-antitoxin system component with homologs in diverse environmental contexts. PLoS One 11: e0158856. DOI: 10.1371/journal.pone.0158856

Wonni I, Cottyn B, Detemmerman L, Dao S, Ouedraogo L, Sarra S, Tekete C, Poussier S, Corral R, Triplett L, Koita O, Koebnik R, Leach J, Szurek B, Maes M, Verdier V (2014). Analysis of Xanthomonas oryzae pv. oryzicola population in Mali and Burkina Faso reveals a high level of genetic and pathogenic diversity. Phytopathology 104: 520-531. DOI: 10.1094/PHYTO-07-13-0213-R

Wu S (2015). Structural and functional characterization of a Xanthomonas type III effector. PhD dissertation. Link: https://vtechworks.lib.vt.edu/handle/10919/73219

Xie XW, Yu J, Xu JL, Zhou YL, Li ZK (2007). Introduction of a non-host gene Rxo1 cloned from maize resistant to rice bacterial leaf streak into rice varieties. Sheng Wu Gong Cheng Xue Bao [Chinese J. Biotechnol.] 23: 607-611. DOI: 10.1016/S1872-2075(07)60039-9

Zhao B, Ardales EY, Raymundo A, Bai J, Trick HN, Leach JE, Hulbert SH (2004). The avrRxo1 gene from the rice pathogen Xanthomonas oryzae pv. oryzicola confers a nonhost defense reaction on maize with resistance gene Rxo1. Mol. Plant Microbe Interact. 17: 771-779. DOI: 10.1094/MPMI.2004.17.7.771

Zhao B, Lin X, Poland J, Trick H, Leach J, Hulbert S (2005). A maize resistance gene functions against bacterial streak disease in rice. Proc. Natl. Acad. Sci. USA 102: 15383-15388. DOI: 10.1073/pnas.0503023102

This fact sheet is based upon work from COST Action CA16107 EuroXanth, supported by COST (European Cooperation in Science and Technology).