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====== The Type III Effector XopL from //Xanthomonas// ======

Author: [[https://www.researchgate.net/profile/Joana_Vicente2|Joana G. Vicente]]\\
Internal reviewer: [[https://www.researchgate.net/profile/Joel_Pothier2|Joël F. Pothier]]\\
Expert reviewer: [[https://www.researchgate.net/profile/Jessica-Erickson-9|Jessica L. Erickson]]

Class: XopL\\
Family: XopL\\
Prototype: XCV3220 (//Xanthomonas euvesicatoria// pv. //euvesicatoria//, ex //Xanthomonas campestris// pv. //vesicatoria//; strain 85-10)\\
GenBank ID: [[https://www.ncbi.nlm.nih.gov/protein/CAJ24951.1|CAJ24951.1]] (660 aa)\\
RefSeq ID: [[https://www.ncbi.nlm.nih.gov/protein/WP_218498620.1|WP_218498620.1]] (560 aa)\\
Examples of other sequences: XopL<sub>Xcc306</sub> [[https://www.ncbi.nlm.nih.gov/protein/21109412|21109412]] (//X. citri// pv. //citri//); XopL<sub>Xcc8004</sub> [[https://www.ncbi.nlm.nih.gov/protein/66575899|66575899]] (//X. campestris// pv. //campestris//)\\
3D structure: [[https://www.rcsb.org/structure/4FC9|4FC9]], [[https://www.rcsb.org/structure/4FCG|4FCG]] (Singer //et al//., 2013). Full-length XopL<sub>Xcv85-10</sub> did not crystallize but fragments XopL[aa 144–450] and XopL[aa 474–660] yielded crystals (Singer //et al//., 2013). The crystal structure of the N-terminal region of XopL showed the presence of a leucine-rich repeat (LRR) domain, that might serve as a protein-protein interaction module for ubiquitination target recognition (Singer //et al//., 2013). The protein represents a new class of E3 ubiquitin ligases.
===== Biological function =====

=== How discovered? ===

XopL was first identified in //X. campestris// pv. //campestris// (//Xcc//) strain 8004 as a candidate T3E due to the presence of a plant-inducible promoter (PIP) box upstream of the CDS, //XC_4273// (Jiang //et al.//, 2009). The CDS //XC_4273//, re-called XopXccLR (LR = leucine-rich repeat) or XopL<sub>Xcc</sub> for the purposes of this article, in //Xcc// 8004 was suggested to be a T3E as it harboured a N-terminal region possessing a translocation signal that was able to target proteins into plant cells (Jiang //et al.//, 2009). It was also shown to be required for //Xcc// proliferation in hosts plant (Jiang //et al.//, 2009). It was only a few years later that the analysis of the genome sequence of //X.// //campestris// pv. //vesicatoria// (//Xcv//) strain 85-10 (synonymous with //X. euvesicatoria// 85-10; //Xe// 85-10) led to the identification of XCV3220 (//xopL<sub>Xe</sub> //) as a new T3E candidate gene and to its more complete characterization (Singer //et al//., 2013). XopL<sub>Xe</sub> was shown to have E3 ubiquitin ligase activity, conferred by a ligase domain with a novel fold (XL-box), capable of interacting with the plant host ubiquitination cascade (Singer //et al.//, 2023).

=== (Experimental) evidence for being a T3E ===

XopL<sub>Xcc</sub> possesses features that are typical of T3Es: the promoter region of the //xopL<sub>Xcc</sub>// gene contains a perfect plant inducible promoter (PIP) box followed by a 10 box similar sequence (TTCGC-N<sub>15</sub>-TTCGC-N<sub>31</sub>-ACGACA) and an LRR motif characteristic of T3Es in pathogenic bacteria (Yan //et al//., 2019). Using an AvrBs1 reporter fusion, XopL<sub>Xcc</sub> was shown to be translocated into plant cells in a //hrpF-// and //hpaB//-dependent manner (Jiang //et al//., 2009). XopL<sub>Xe</sub> also contains a PIP box (plant inducible promoter) in its promoter (TTCG-N<sub>16</sub>-TTCG; genome position 3669238-261) and co-regulation with the T3S system was confirmed by RT-PCR (Singer //et al.//, 2013). Type III-dependent secretion and translocation was confirmed by //in vitro//secretion and //in vivo//translocation assays (Singer //et al.//, 2013).
=== Regulation ===

The //xopL// <sub>Xcc8004</sub> 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//), including //xopL//, were significantly reduced in the //Xanthomonas oryzae// pv. //oryzae// Δ//xrvC// mutant compared with those in the wild-type strain PXO99<sup>A</sup>  (Liu //et al.//, 2016).

The expression of //xopL<sub>Xcc</sub>// gene is positively regulated by HrpG/HrpX (Yan //et al//., 2019).
=== Phenotypes ===

  * XopL<sub>//Xe//</sub>  from //X. euvesicatoria//  85-10 (previously referred to as //X. campestris// pv. //euvesicatoria//; //Xcv//) displays E3 ubiquitin ligase activity (Singer //et al.//, 2013).
  * XopL//<sub>Xe</sub>// inhibits expression of the elf18- and flg22-induced defense gene pNHL10 in //Arabidopsis// mesophyll protoplasts independent of E3 ligase function (Singer //et al.//, 2013).
  * XopL//<sub>Xe</sub>// suppresses ABA responsive reporter //pRD29b:GUS// and PTI reporter //pFRK1:LUC// in //Arabidopsis// protoplasts (Popov //et al//., 2016).
  * XopL//<sub>Xcc</sub>// interferes with innate immunity (Yan //et al.//, 2019; Huang //et al.//, 2024a) and SA signaling in //Arabidopsis// (Huang //et al.//, 2024).
  * XopL//<sub>Xe</sub>// triggers cell death in //Nicotiana benthamiana// in an E3 ligase dependant manner (Singer //et al.//, 2013). XopL<sub>Xoc</sub> from //X. oryzae// pv. //oryzicola// and XopL<sub>Xoo</sub> from //X. oryzae// pv. //oryzae// PX099A also cause cell death in this model (Ma //et al.//, 2020).
  * Distantly related XopL homolog XopL//<sub>Xcc</sub>// from //Xcc// 8004 failed to cause plant cell death. (Ortmann //et al.//, 2023).
  * XopL//<sub>Xcc</sub>// is required for full virulence and growth of //Xcc// 8004 in the host plant Chinese radish (Jiang //et al.//, 2009) but not in //Arabidopsis// (Huang et al, 2024). However a mutant for 17 effectors (Δ//17E//) supplemented with //xopL<sub>Xcc</sub> // did grow better than Δ//17E//.
  * XopL//<sub>Xcc</sub>// constitutive overexpression in //Arabidopsis// led to enhanced //Xcc// 8004 virulence and suppressed callose deposition and oxidative burst (Huang //et al.//, 2024).
  * XopL//<sub>Xe</sub>// is required for full virulence of //Xe// 85-10 on tomato (Leong et al., 2022).
  * XopL//<sub>Xap</sub>// supports //X. axonopodis //pv.// punicae //multiplication in pomegranate by suppressing plant immune responses including plant cell death (Soni //et al//., 2017).
  * Transient expression of XopL//<sub>Xe</sub> //, led to a nearly complete elimination of stromules and the relocation of plastids to the nucleus and further characterization revealed that the E3 ligase activity is essential for the two plastid phenotypes (//Erickson et al//., 2018).
  * //Xe //85-10 suppresses host autophagy by utilizing type-III effector XopL//<sub>Xe</sub> //. Intriguingly, XopL//<sub>Xe</sub>// is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery (Leong// et al.//, 2022).
=== Localization ===

Several localization patterns have been reported for XopL proteins in epidermal cells with some strain dependent differences. XopLs are most often tagged at the C-terminus, with the exception of the studies by Leong //et al//., 2022 and Yan //et al.//, 2019.

  * __Cytosolic localization__ has been reported for XopL//<sub>Xe</sub>// (Erickson //et al.//, 2018), XopL//<sub>Xcc</sub>// (Yan //et al//., 2019; Ortmann //et al//. 2023), XopL<sub>//Xoo//</sub> (Ma //et al//., 2020; Ortmann //et al//., 2023), and XopL//<sub>Xac</sub> //  from //X. citri// pv. //citri// (Ortmann et al. 2023) in //N. benthamiana//.
  * __Nuclear localization__ was reported for XopL//<sub>Xe</sub>//, XopL<sub>//Xcc//</sub> (Yan //et al.//, 2019; Ortmann //et al//., 2023) and XopL//<sub>Xac</sub>// in //N. benthamiana// (Ortmann et al. 2023), but not for XopL//<sub>Xoo</sub>// in //N. benthamiana// or XopL//<sub>Xap</sub>// from //X. axonopodis// pv. //punicae// in //Arabidopsis// protoplasts (Soni //et al.//, 2017; Ortmann //et al.,// 2023).
  * __Plasma membrane localization__ has been reported for XopL<sub>//Xap//</sub> transiently expressed in //N. benthmiana// (Soni //et al//., 2017) and XopL//<sub>Xcc</sub>// expressed in //Arabidopsis// protoplasts (Huang //et al//. 2024b; Yan //et al.// , 2019) and //N. benthamiana// leaves (Yan //et al//., 2019).
  * __Microtubule localization__ has been reported for XopL//<sub>Xe</sub>//, XopL<sub>//Xoo//</sub> and XopL//<sub>Xac</sub>//, whereas the distantly related XopL//<sub>Xcc</sub>// failed to localize to microtubules in //N. benthamiana// (Ortmann //et al.//, 2023).
  * __Autophagosome localization__ has been reported for XopL//<sub>Xe</sub>// (co-localizes with autophagy markers RFP-ATG8e and SH3P2-RFP; Leong //et al//., 2022).
=== Enzymatic function ===

XopL<sub>Xe</sub> harbours an unstructured N-terminus, followed by three alpha helices, an leucine-rich repeat (LRR) domain and an XL-box. Mutation of amino acids in the central cavity of the XL-box disrupt E3 ligase activity and prevent XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggest that thioester-linked ubiquitin-E3 ligase intermediates are not formed during XopL-mediated ubiquitination. Suppression of PAMP responses solely depends on the N-terminal LRR domain (Singer //et al//., 2013), while microtubule binding relies on a proline-rich region in the unstructured region and the alpha-helical region (Ortman //et al//., 2023).

=== Interaction partners ===

XopL<sub>Xe</sub> interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection (Leong //et al.//, 2022). XopL<sub>Xoo</sub> interacts with and degrades ferredoxin from //N. benthamiana//, part of the electron transport chain (Ma //et al.//, 2020). XopLXcc interaction with proton pump interactor 1 (PPI1) in //Arabidopsis// (Huang //et al//., 2024b).

===== Conservation =====

=== In xanthomonads ===

Yes (//e.g.//, //X. euvesicatoria//, //X. citri//, //X. axonopodis//, //X. oryzae//, //X. oryzicola//, //X//. //fragariae//, //X//. //perforans, X. gardneri//, //X. campestris// pv. //campestris//, but not //X. campestris// pv. //raphani//, in some //X. arboricola// pathovars). See for example [[https://doi.org/10.1094/MPMI-22-11-1401|Table 2]] in Jiang //et al//. (2009) and [[https://doi.org/10.1371/journal.ppat.1003121.s001|Figure S1]] in Singer //et al//. (2013).

=== In other plant pathogens/symbionts ===

No.

===== 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: [[https://d-nb.info/1116951061/34|d-nb.info/1116951061/34]]FIXME

Erickson JL, Adlung N, Lampe C, Bonas U, Schattat MH (2018). The //Xanthomonas// effector XopL uncovers the role of microtubules in stromule extension and dynamics in //Nicotiana benthamiana//. Plant J. 93: 856-870. DOI:[[https://doi.org/10.1111/tpj.13813|10.1111/tpj.13813]]

Huang J, Dong Y, Li N, He Y, Zhou H (2024a). The type III effector XopL//<sub>Xcc</sub>// in //Xanthomonas campestris// pv. //campestris// targets the proton pump interactor 1 and suppresses innate immunity in //Arabidopsis//. Int. J. Mol. Sci. 25: 9175. DOI: [[https://doi.org/10.3390/ijms25179175|10.3390/ijms25179175]]

Huang J, Zhou H, Zhou M, Li N, Jiang B, He Y (2024b). Functional analysis of type III effectors in //Xanthomonas campestris// pv. //campestris// reveals distinct roles in modulating //Arabidopsis// innate immunity. Pathogens 13: 448. DOI: [[https://doi.org/10.3390/pathogens13060448|10.3390/pathogens13060448]]

Jiang W, Jiang BL, Xu RQ, Huang JD, Wei HY, 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: [[https://doi.org/10.1094/MPMI-22-11-1401|10.1094/MPMI-22-11-1401]]

Leong JX, Raffeiner M, Spinti D, Langin G, Franz-Wachtel M, Guzman AR, Kim JG, Pandey P, Minina AE, Macek B, Hafrén A, Bozkurt TO, Mudgett MB, Börnke F, Hofius D, Üstün S (2022). A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component. EMBO J. 41: e110352. DOI: [[https://doi.org/10.15252/embj.2021110352|10.15252/embj.2021110352]]

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: [[https://doi.org/10.1093/femsle/fnw067|10.1093/femsle/fnw067]]

Ma W, Xu X, Cai L, Cao Y, Haq F, Alfano JR, Zu B, Zou L, Chen G (2020). A //Xanthomonas oryzae// type III effector XopL causes cell death through mediating ferredoxin degradation in //Nicotiana benthamiana//. Phytopathol Res. 2: 16. DOI: 10.1186/s42483-020-00055-w

Ortmann S, Marx J, Lampe C, Handrick V, Ehnert TM, Zinecker S, Reimers M, Bonas U, Erickson JL (2023). A conserved microtubule-binding region in //Xanthomonas// XopL is indispensable for induced plant cell death reactions. PLoS Pathog. 19: e1011263. DOI: [[https://doi.org/10.1371/journal.ppat.1011263|10.1371/journal.ppat.1011263]]

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: [[https://doi.org/10.1094/MPMI-07-16-0137-R|10.1094/MPMI-07-16-0137-R]]

Singer AU, Schulze S, Skarina T, Xu X, Cui H, Eschen-Lippold L, Egler M, Srikumar T, Raught B, Lee J, Scheel D, Savchenko A, Bonas U (2013). A pathogen type III effector with a novel E3 ubiquitin ligase architecture. PLoS Pathog. 9: e1003121. DOI: [[https://doi.org/10.1371/journal.ppat.1003121|10.1371/journal.ppat.1003121]]

Soni M, Mondal KK. (2017). //Xanthomonas axonopodis// pv. //punicae// employs XopL effector to suppress pomegranate immunity. J. Integr. Plant Biol. 60: 341-357. DOI: [[https://doi.org/10.1111/jipb.12615|10.1111/jipb.12615]]

Yan X, Tao J, Luo HL, Tan LT, Rong W, Li HP, He CZ (2019). A type III effector XopL<sub>Xcc8004</sub> is vital for //Xanthomonas campestris// pathovar //campestris// to regulate plant immunity. Res. Microbiol. 170: 138-146. DOI: [[https://doi.org/10.1016/j.resmic.2018.12.001|10.1016/j.resmic.2018.12.001]]

===== Acknowledgements =====

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