The Type III Effector XopQ from //Xanthomonas//

Author: Valérie Olivier & Tamara Popović
Internal reviewer: Ralf Koebnik

Class: XopQ
Family: XopQ
Prototype: XopQ (Xanthomonas euvesicatoria pv. euvesicatoria, ex Xanthomonas campestris pv. vesicatoria; strain 85-10)
GenBank ID: AAV74206.1 (464 aa)
RefSeq ID: WP_011349176.1 (464 aa)
3D structure: 4KL0 (Yu et al., 2013); 4P5F (Yu et al., 2014); 7JLU, 7JLV, 7JLX (Martin et al., 2020)

XopQ was identified in a genetic screen, using a Tn5-based transposon construct harboring the coding sequence for the HR-inducing domain of AvrBs2, but devoid of the effectors' T3SS signal, that was randomly inserted into the genome of X. campestris pv. vesicatoria (Xcv)strain 85-10. The XopQ::AvrBs2 fusion protein triggered a Bs2-dependent hypersensitive response (HR) in pepper leaves (Roden et al., 2004). XopQ was also identified in X. campestris pv. campestris (Xcc) strain 8004 as a candidate T3E due to the presence of a plant-inducible promoter (PIP) box in its gene, XC_3177 (Jiang et al., 2009).

Type III-dependent secretion was confirmed using a calmodulin-dependent adenylate cyclase reporter assay, with a ΔhrpF mutant strain serving as negative control (Roden et al., 2004). Using an AvrBs1 reporter fusion, XopQ_Xcc8004 was shown to be translated into plant cells in a hrpF- and hpaB-dependent manner Jiang et al., 2009).

The xopQ _Xcc8004 gene contains a PIP box and was shown to be controlled by hrpG and hrpX (Jiang et al., 2009).

qRT-PCR revealed that transcript levels of 15 out of 18 tested non-TAL effector genes (as well as the regulatory genes hrpG and hrpX) were significantly reduced in the Xanthomonas oryzae pv. oryzae ΔxrvC mutant compared with those in the wild-type strain PXO99^A, but this did not apply to xopQ (Liu et al., 2016).

• Roden et al. did not find significant growth defects of a Xcv ΔxopQ mutant in susceptible pepper and tomato leaves (Roden et al., 2004).
• XopQ_Xcc8004 is required for full virulence and growth of X. campestris pv. campestris in the host plant Chinese radish (Jiang et al., 2009).
• In X. oryzae pv. oryzae (Xoo), XopQ was described by Sinha et al. to suppress DAMP-induced PTI in rice. Indeed, Xoo secretes hydrolytic enzymes such as LipA (Lipase/Esterase) that damage rice cell walls and induce innate immune responses. XopQ was found to suppress LipA-induced innate immune responses in rice (Sinha et al., 2013).
• XopQ_Xcv suppresses cell death controlled by components of the MAP kinase cascade MAPKKKα/MEK2/SIPK and induced by certain R/avr gene pairs, such as Pto/avrPto and Gpa2/RBP–1, but not that induced by other gene pairs (Bs3/avrBs3, RPS2/avrRpt2, C9/avr9, Rx2/Cp) (Teper et al., 2014).
• Consistent with a role in ETI, TFT4 mRNA abundance increased during the incompatible interaction of tomato and pepper with Xcv (Teper et al., 2014).
• Mutations of two potential active site residues, D116 and Y279, resulted in Xoo mutants with reduced virulence on rice and reduced hypersensitive response (HR) on Nicotiana benthamiana, a nonhost. However, Arabidopsis lines expressing either xopQ or xopQ _Y279A were equally proficient at suppression of LipA-induced callose deposition (Gupta et al., 2015).
• Compatibility studies with X. euvesicatoria pv. perforans revealed that a double deletion of avrBsT and xopQ allows a host range expansion for Nicotiana benthamiana (Schwartz et al., 2015).
• The avirulence activity of XopQ derivatives did not correlate with macroscopically visible plant reactions upon transient expression in N. benthamiana. It was therefore speculated that N. benthamiana might encode two resistance proteins for the recognition of XopQ (Adlung, 2016).
• Transient co-expression of XopQ::GFP and XopS::GFP in N. benthamiana triggered cell death reactions, which were not observed when each effector was expressed alone. Bimolecular fluorescence complementation using split-YFP derivatives revealed that XopQ and XopS co-localize in the nucleus. These results suggested that both effectors may form a protein-protein complex in planta (Adlung, 2016).
• XopQ suppressed cell death reactions in N. benthamiana that were triggered by three Xcv type III effectors (XopB, XopJ, XopL), whereas cell death reactions triggered by AvrBsT were not suppressed by XopQ (Adlung, 2016).
• XopQ-mediated cell death suppression in N. benthamiana during transient expression assays was later shown to result from an attenuation of Agrobacterium‐mediated protein expression rather than reflecting a genuine XopQ virulence activity (Adlung & Bonas, 2017).
• A ΔxopN–ΔxopQ double knock-out mutant in X. phaseoli pv. manihotis (Xpm) was less aggressive in the cassava host plant than its single mutation counterparts. In addition, in planta bacterial growth was reduced at 5 dpi in the double mutant with respect to the wild-type strain CIO151 and individual knock-out strains. The phenotype of the double mutant could be complemented when transforming a plasmid containing xopQ. These results confirmed that xopN/ /and xopQ are functionally redundant in Xpm (Medina et al., 2017). * A reverse genetics screen identified Recognition of XopQ 1 (Roq1), a nucleotide-binding leucine-rich repeat (NLR) protein with a Toll-like interleukin-1 receptor (TIR) domain, which mediates XopQ recognition in N. benthamiana. Roq1 orthologs appear to be present only in the Nicotiana genus. Expression of Roq1 was found to be sufficient for XopQ recognition in both the closely-related Nicotiana sylvestris and the distantly-related beet plant (Beta vulgaris) (Schultink et al., 2017). * Roq1 is able to recognize XopQ alleles from various Xanthomonas species, as well as HopQ1 from Pseudomonas, demonstrating widespread potential application in protecting crop plants from these pathogens (Schultink et al., 2017). * The coiled-coil NLR protein N requirement gene 1 (NRG) interacts with EDS1 and acts downstream of Roq1 and EDS1 to mediate XopQ/HopQ1-triggered ETI. In addition, Roq1, EDS1, and NRG1 mediate XopQ-triggered transcriptional changes in N. benthamiana and regulate resistance to Xanthomonas and Pseudomonas species that carry the effectors XopQ or HopQ1. This study suggests that NRG1 may be a conserved key component in TNL-mediated signaling pathways (Qi et al., 2018). * Roq1 is also involved in the recognition of RipB, the homolog of XopQ in Ralstonia solanacearum: The RipB‐induced resistance against R. solanacearum was abolished in Roq1‐silenced plants (Nakano & Mukaihara, 2019). * Effectors that interact with 14–3–3 proteins may provide plant-pathogenic bacteria with the ability to modulate PTI as well as ETI. Suppression of immune responses induced by a xopN–xopQ–xopX–xopZ quadruple mutant by the XopQ effector may be both suppression of ETI as well as suppression of DTI (damage-triggered immunity) caused by the release of DAMPs by the quadruple mutant strain (Deb et al., 2019). * Roq1 was found to confer immunity to Xanthomonas (containing XopQ), P. syringae (containing the XopQ homolog HopQ1), and Ralstonia (containing the XopQ homolog RipB) when expressed in tomato (Thomas et al., 2020). * Strong resistance to Xanthomonas euvesicatoria pv. perforans was observed with transgenic tomato plants expressing Roq1 from N. benthamiana in three seasons of field trials with both natural and artificial inoculation. The Roq1 gene can therefore be used to provide safe, economical, and effective control of these pathogens in tomato and other crop species and reduce or eliminate the need for traditional chemical controls (Thomas et al., 2020). * Agrobacterium-mediated transient expression of both XopQ and XopX in rice cells resulted in induction of rice immune responses. These immune responses were not observed when either protein was individually expressed in rice cells. XopQ-XopX induced rice immune responses were not observed with a XopX mutant that is defective in 14-3-3 binding (Deb et al., 2020). * A screen for Xanthomonas effectors which can suppress XopQ-XopX induced rice immune responses, led to the identification of five effectors, namely XopU, XopV, XopP, XopG and AvrBs2, that could individually suppress these immune responses. These results suggest a complex interplay of Xanthomonas T3SS effectors in suppression of both pathogen-triggered immunity and effector-triggered immunity to promote virulence on rice (Deb et al., 2020). * Expression of XopQ in In N. benthamiana triggers Roq1-dependent effector-triggered immunity (ETI) responses accompanied by the accumulation of plastids around the nucleus and the formation of stromules. It was shown that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI and that stromule formation, in contrast to chloroplast perinuclear dynamics, is an integral part of the N. benthamiana ETI response, where both NRG1 and ADR1 play a role in this ETI response (Prautsch et al., 2023). === Localization === Cytoplasma and nucleus (Deb et al., 2019). === Enzymatic function === XopQ is structurally homologous to an inosine-uridine nucleoside N-ribohydrolase from a protazoan parasite, as shown be 3D-PSSM analysis. Such proteins are implicated in the ability of many organisms to salvage nucleotides from their environment. Like other homologs, XopQ contains conserved aspartate residues found in the active site of the enzyme. It was speculated that XopQ may function as a scavenging hydrolase in planta or may interfere with plant cell processes by binding and sequestering nucleosides important for plant signaling and/or metabolism (Roden et al., 2004). Despite such similarities, later structural and functional studies revealed that XopQ_Xoo does not exhibit the expected activity of a nucleoside hydrolase (Yu et al., 2013). Purified XopQ_Xoo did not show NH activity on standard nucleoside substrates but exhibited ribose hydrolase activity on the nucleoside substrate analogue 4-nitrophenyl β-D-ribofuranoside (Gupta et al., 2015). The D116A and Y279A mutations cause a reduction in biochemical activity (Gupta et al., 2015). In 2014, Yu et al. reported the crystal structure of XopQ_Xoo in complex with adenosine diphosphate ribose (ADPR), which is involved in regulating cytoplasmic Ca²⁺ concentrations in eukaryotic cells, which is one of the key events in the immune response elicited by pathogen invasion of a host plant (Yu et al., 2014). ADPR is bound to the active site of XopQ_Xoo with its ribosyl end tethered to the Ca²⁺ coordination shell. The binding of ADPR is further stabilized by interactions mediated by hydrophobic residues that undergo ligand-induced conformational changes. XopQ_Xoo is capable of binding a novel chemical bearing a ribosyl moiety (Yu et al., 2014). === Interaction partners === Using protein-protein interaction studies in yeast and in planta, XopQ_Xcv was shown to physically interacts with the 14–3–3 protein TFT4 from tomato (Solanum lycopersicum) (Teper et al., 2014). A mutation in the putative 14–3–3 binding site of XopQ (S65A) impaired interaction of the effector with TFT4 from pepper and tomato (Capsicum annuum) and its virulence function in planta (Teper et al., 2014). Yeast 2-hybrid assays revealed that XopQ_Xcv interacts with multiple, but perhaps not all 14–3–3 protein isoforms (Teper et al., 2014; Dubrov et al., 2018). Bimolecular fluorescence complementation assays upon transient expression in N. benthamiana using split-YFP derivatives revealed that XopQ may interact with itself and also with XopS, maybe forming a large protein complex in planta (Adlung, 2016). Roq1 (Recognition of XopQ), a nucleotide-binding leucine-rich repeat (NLR) protein with a Toll-like interleukin-1 receptor (TIR) domain, was found to co-immunoprecipitate with XopQ, for the first time suggesting a physical association between the two proteins (Schultink et al., 2017). Structural biology later demonstrated that ROQ1 directly binds to both the predicted active site and surface residues of XopQ while forming a tetrameric resistosome complex that brings together the TIR domains for downstream immune signaling (Martin et al., 2020). XopQ_Xoo was shown to interact in yeast and in planta with two rice 14–3–3 proteins, Gf14f and Gf14g (Deb et al., 2019). A serine to alanine mutation (S65A) of a 14–3–3 interaction motif in XopQ abolished the ability of XopQ to interact with the two 14–3–3 proteins and to suppress innate immunity. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-IP assays indicated that XopQ and XopX interact with each other (Deb et al., 2020). ===== Conservation ===== === In xanthomonads === XopQ is a widely conserved across Xanthomonas ssp., such as X. campestris,X. citri, X. euvesicatoria, X. oryzae (Roden et al., 2004; Furutani et al., 2009; Hajri et al., 2009; Thomas et al., 2020). Since the G+C content of the xopQ gene is similar to that of the Xcvhrp gene cluster, it may be a member of a “core” group of Xanthomonas spp. effectors (Roden et al., 2004). === In other plant pathogens/symbionts === XopQ shares homology with the Ralstonia solanacearum effector RipB and the Pseudomonas syringae pv. tomato effector HolPtoQ/HopQ (Roden et al., 2004; Büttner & Bonas, 2010). Unlike most recognized effectors, alleles of XopQ/HopQ1 are highly conserved and present in most plant-pathogenic strains of Xanthomonas and P. syringae, and the homolog of XopQ/HopQ1, named RipB, is present in most Ralstonia strains (Thomas et al., 2020). ===== References ===== Adlung N (2016). Charakterisierung der Avirulenzaktivität von XopQ und Identifizierung möglicher Interaktoren von XopL aus Xanthomonas campestris pv. vesicatoria. Doctoral Thesis. Martin-Luther-Universität Halle-Wittenberg, Germany. PDF: d-nb.info/1116951061/34 Adlung N, Bonas U (2017). 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Xanthomonas oryzae pv. oryzae XopQ protein suppresses rice immune responses through interaction with two 14-3-3 proteins but its phospho-null mutant induces rice immune responses and interacts with another 14-3-3 protein. Mol. Plant Pathol. 20: 976-989. DOI: 10.1111/mpp.12807 Dubrow Z, Sunitha S, Kim JG, Aakre CD, Girija AM, Sobol G, Teper D, Chen YC, Ozbaki-Yagan N, Vance H, Sessa G, Mudgett MB (2018). Tomato 14-3-3 proteins are required for Xv3 disease resistance and interact with a subset of Xanthomonas euvesicatoria effectors. Mol. Plant Microbe Interact. 31: 1301-1311. DOI: 10.1094/MPMI-02-18-0048-R Furutani A,Takaoka M, Sanada H, Noguchi Y, Oku T, Tsuno K, Ochiai H, Tsuge S (2009). Identification of novel type III secretion effectors in Xanthomonas oryzae pv. oryzae. Mol. Plant Microbe Interact. 22: 96-106. DOI: 10.1094/MPMI-22-1-0096 Gupta MK, Nathawat R, Sinha D, Haque AS, Sankaranarayanan R, Sonti RV (2015). Mutations in the predicted active site of Xanthomonas oryzae pv. oryzae XopQ differentially affect virulence, suppression of host innate immunity, and induction of the HR in a nonhost plant. Mol. Plant Microbe Interact. 28:195-206. DOI: 10.1094/MPMI-09-14-0288-R Hajri A, Brin C, Hunault G, Lardeux F, Lemaire C, Manceau C, Boureau T, Poussier S (2009). A “repertoire for repertoire” hypothesis: repertoires of type three effectors are candidate determinants of host specificity in Xanthomonas. PLoS One 4: e6632. DOI: 10.1371/journal.pone.0006632 Jiang W, Jiang B, Xu R, Huang J, Wei H, Jiang GF, Cen WJ, Liu J, Ge YY, Li GH, Su LL, Hang XH, Tang DJ, Lu GT, Feng JX, He YQ, Tang JL (2009). Identification of six type III effector genes with the PIP box in Xanthomonas campestris pv. campestris and five of them contribute individually to full pathogenicity. Mol. Plant Microbe Interact. 22: 1401-1411. DOI: 10.1094/MPMI-22-11-1401 Liu Y, Long J, Shen D, Song C (2016). Xanthomonas oryzae pv. oryzae requires H-NS-family protein XrvC to regulate virulence during rice infection. FEMS Microbiol. Lett. 363: fnw067. DOI: 10.1093/femsle/fnw067 Martin R, Qi T, Zhang H, Liu F, King M, Toth C, Nogales E, Staskawicz BJ (2020). Structure of the activated ROQ1 resistosome directly recognizing the pathogen effector XopQ. Science 370: eabd9993. DOI: 10.1126/science.abd9993 Medina CA, Reyes PA, Trujillo CA, Gonzalez JL, Bejarano DA, Montenegro NA, Jacobs JM, Joe A, Restrepo S, Alfano JR, Bernal A (2018). The role of type III effectors from Xanthomonas axonopodis pv. manihotis in virulence and suppression of plant immunity. Mol. Plant Pathol. 19: 593-606. DOI: 10.1111/mpp.12545 Nakano M, Mukaihara T (2019). The type III effector RipB from Ralstonia solanacearum RS1000 acts as a major avirulence factor in Nicotiana benthamiana and other Nicotiana species. Mol. Plant Pathol. 20: 1237-1251. DOI: 10.1111/mpp.12824 Prautsch J, Erickson JL, Özyürek S, Gormanns R, Franke L, Lu Y, Marx J, Niemeyer F, Parker JE, Stuttmann J, Schattat MH (2023). Effector XopQ-induced stromule formation in Nicotiana benthamiana depends on ETI signaling components ADR1 and NRG1. Plant Physiol. 191: 161-176. DOI: 10.1093/plphys/kiac481 Qi T, Seong K, Thomazella DPT, Kim JR, Pham J, Seo E, Cho MJ, Schultink A, Staskawicz BJ (2018). NRG1 functions downstream of EDS1 to regulate TIR-NLR-mediated plant immunity in Nicotiana benthamiana. Proc. Natl. Acad. Sci. USA 115: E10979-E10987. DOI: 10.1073/pnas.1814856115 Roden JA, Belt B, Ross JB, Tachibana T, Vargas J, Mudgett MB (2004). A genetic screen to isolate type III effectors translocated into pepper cells during Xanthomonas infection. Proc. Natl. Acad. Sci. USA 101: 16624-16629. DOI: 10.1073/pnas.0407383101 Schultink A, Qi T, Lee A, Steinbrenner AD, Staskawicz B (2017). Roq1 mediates recognition of the Xanthomonas and Pseudomonas effector proteins XopQ and HopQ1. Plant J. 92: 787-795. DOI: 10.1111/tpj.13715 Schwartz AR, Potnis N, Timilsina S, Wilson M, Patané J, Martins J Jr, Minsavage GV, Dahlbeck D, Akhunova A, Almeida N, Vallad GE, Barak JD, White FF, Miller SA, Ritchie D, Goss E, Bart RS, Setubal JC, Jones JB, Staskawicz BJ (2015). Phylogenomics of Xanthomonas field strains infecting pepper and tomato reveals diversity in effector repertoires and identifies determinants of host specificity. Front. Microbiol. 6: 535. DOI: 10.3389/fmicb.2015.00535 Sinha D, Gupta MK, Patel HK, Ranjan A, Sonti RV (2013). Cell wall degrading enzyme induced rice innate immune responses are suppressed by the type 3 secretion system effectors XopN, XopQ, XopX and XopZ of Xanthomonas oryzae pv. oryzae. PLoS One 8: e75867. DOI: 10.1371/journal.pone.0075867 Teper D, SalomonD, Sunitha S, Kim JG, Mudgett MB, Sessa G. (2014). Xanthomonas euvesicatoria type III effector XopQ interacts with tomato and pepper 14-3-3 isoforms to suppress effector-triggered immunity. Plant J. 77: 297-309. DOI: 10.1111/tpj.12391 Thomas NC, Hendrich CG, Gill US, Allen C, Hutton SF, Schultink A (2020). The immune receptor Roq1 confers resistance to the bacterial pathogens Xanthomonas, Pseudomonas syringae, and Ralstonia in tomato. Front. Plant Sci. 11: 463. DOI: 10.3389/fpls.2020.00463 Yu S, Hwang I, Rhee S (2013). Crystal structure of the effector protein XOO4466 from Xanthomonas oryzae. J. Struct. Biol. 184: 361-366. DOI: 10.1016/j.jsb.2013.08.007 Yu S, Hwang I, Rhee S (2014). The crystal structure of type III effector protein XopQ from Xanthomonas oryzae complexed with adenosine diphosphate ribose. Proteins 82: 2910-2914. DOI: 10.1002/prot.24656 ===== Acknowledgements ===== This fact sheet is based upon work from COST Action CA16107 EuroXanth, supported by COST (European Cooperation in Science and Technology).