The Type III Effector XopN from //Xanthomonas//

Author: Jakub Pečenka
Internal reviewer: Joana G. Vicente

Class: XopN
Family: XopN
Prototype: XopN (Xanthomonas euvesicatoria pv. euvesicatoria, ex Xanthomonas campestris pv. vesicatoria; strain 85-10)
GenBank ID: AAV74202.1 (733 aa)
RefSeq ID: WP_011348010.1 (733 aa)
3D structure: unknown - similar to phosphatase 2a (pr65/A) (Roden et al., 2004).

XopN 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 XopN::AvrBs2 fusion protein triggered a Bs2-dependent hypersensitive response (HR) in pepper leaves (Roden et al., 2004).

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).

Start codon of xopN was found downstream of a conserved cis-regulatory element, the plant-inducible promoter (PIP) box (TTCGG-N15-TTCTG). xopN is regulated by hrpX and hrpG genes (Jiang et al., 2008; Cheong et al., 2013).

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 xopN (Liu et al., 2016).

• XopN_Xcv is required for the pathogens' maximal growth in the leaf tissue of tomato and pepper plants (Roden et al., 2004).
• Its homolog XopN _Xcc was found as well to be required for full virulence on Chinese radish (Jiang et al., 2008).
• XopN has been shown to play a role in host defence systems causing the reduction of PAMP-triggered immune responses and reduce the callose deposition in the host tissue. Moreover the deletion of xopN open reading frame (ORF) reduced the Xcv strain virulence exhibited by lower bacterial spot symptoms occurrence (Kim et al., 2009).
• The role of XopN in X. oryzae pv. oryzae is dependent on leaf stage (Cheong et al., 2013).
• XopN has been shown to be required for maximal pathogenicity of X. axonopodis pv. punicae (Xap) in pomegranate (Kumar and Mondal, 2013). The deletion of XopN from Xap caused higher accumulation of reactive oxygen species showing that XopN suppresses ROS-mediated defense responses during blight pathogenesis in pomegranate (Kumar et al., 2016).
• 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).
• Agrobacterium mediated transient transfer of the gene for XopN resulted in suppression of rice innate immune responses induced by LipA, a hydrolitic enzyme secreted by X. oryzae pv. oryzae (Xoo), but a xopN ^- mutant of Xooretains the ability to suppress these innate immune responses indicating other functionally redundant proteins; XopQ, XopX and XopZ were shown to be suppressors of LipA induced innate immune responses; mutation in any one of the xopN, xopQ, xopX or xopZ genes causes partial virulence deficiency (Sinha et al., 2013). XopN was shown to contribute significantly to X. oryzae pv. oryzae (Xoo) virulence on a susceptible rice variety Nipponbare. XopN was shown to be highly translocated to suppress rice defense responses (Mo et al., 2020).
• XopN and AvrBS2 were shown to significantly contribute to virulence of X. oryzae pv. oryzicola (Xoc GX01) (Liao et al., 2020).

XopN was localized by confocal microscopy using fluorescent tagged fusion (yellow fluorescent protein [YFP]-XopN). [YFP]-XopN was localized throughout the plant cytoplasm and also associated with the plant plasma membrane (PM) (Kim et al., 2009). Kumar et al. (2016) demonstrated that XopN is localized in the pasma membrane of N. benthamiana, pomegranate and onion cells.

XopN binds TARK1, a tomato atypical receptor kinase required for PTI. Taylor et al. (2012) showed that XopN promotes TARK1/TFT1 complex formation in vitro and in planta by functioning as a molecular scaffold.TFT proteins are involved in immune signaling during X. euvesicatoria infection and can interact with multiple effectors including XopN (Dubrow et al., 2018). TARK1 was shown to interact with proteins predicted to be associated with stomatal closure (Guzman et al., 2020).

Three effectors (XopZ, XopN and XopV) were shown to be able to supress the peptidoglycan-triggered MAPK activation and a triple mutant of Xoo lacking these genes showed additively reduced virulence (Long et al., 2018).

XopN interact with two types of proteins in tomato: Tomato Atypical Receptor-like Kinase1 (TARK1) and four Tomato Fourteen-Three-Three isoforms (TFT1, TFT3, TFT5, and TFT6) (Kim et al., 2009). XopN interacts with the tomato 14-3-3 isoform TFT1 that functions in PTI and is a XopN virulence target (Taylor et al., 2012).

Two rice proteins, OsVOZ2 and a putative thiamine synthase (OsXNP) were identified as targets of XopN_KXO85 by yeast two-hybrid screening (Cheong et al., 2012).

Yes (e.g., X. axonopodis, X. campestris, X. citri, X. oryzae). Since the G+C content of the xopN gene is similar to that of the Xcv hrp gene cluster, it may be a member of a “core” group of Xanthomonas spp. effectors (Roden et al., 2004).

Yes (e.g., Pseudomonas spp.) (Kim et al., 2009).

Cheong H, Kim CY, Jeon JS, Lee BM, Sun Moon J, Hwang I (2013). Xanthomonas oryzae pv. oryzae type III effector XopN targets OsVOZ2 and a putative thiamine synthase as a virulence factor in rice. PloS ONE 8: e73346. DOI: 10.1371/journal.pone.0073346.

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

Guzman AR, Kim JG, Taylor KW, Lanver D, Mudgett MB (2020). Tomato Atypical Receptor Kinase1 is involved in the regulation of preinvasion defense. Plant Physiol. 183: 1306-1318. DOI: 10.1104/pp.19.01400

Jiang B, He Y, Cen W, Wei H, Jiang G, Jiang W, Hang X, Feng J, Lu G, Tang D, Tang J (2008). The type III secretion effector XopXccN of Xanthomonas campestris pv. campestris is required for full virulence. Res. Microbiol. 159: 216-220. DOI: 10.1016/j.resmic.2007.12.004

Kim JG, Li X, Roden JA, Taylor KW, Aakre CD, Su B, Landone S, Kirik A, Chen Y, Baranage G, Martin BG, Mudgett BM, McLane H (2009). Xanthomonas T3S effector XopN suppresses PAMP-triggered immunity and interacts with a tomato atypical receptor-like kinase and TFT1. Plant Cell 21: 1305-1323. DOI: 10.1105/tpc.108.063123

Kumar R, Mondal KK (2013). XopN-T3SS effector modulates in planta growth of Xanthomonas axonopodis pv. punicae and cell-wall-associated immune response to induce bacterial blight in pomegranate. Physiol. Mol. Plant Pathol. 84: 36-43. DOI: 10.1016/j.pmpp.2013.06.002

Kumar R, Soni M, Mondal KK (2016). XopN-T3SS effector of Xanthomonas axonopodis pv. punicae localizes to the plasma membrane and modulates ROS accumulation events during blight pathogenesis in pomegranate. Microbiol. Res. 193: 111-120. DOI: 10.1016/j.micres.2016.10.001

Liao ZX, Li JY, Mo XY, Ni Z, Jiang W, He YQ, Huang S (2020). Type III effectors xopN and avrBS2 contribute to the virulence of Xanthomonas oryzae pv. oryzicola strain GX01. Res. Microbiol. 171: 102-106. DOI: 10.1016/j.resmic.2019.10.002

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

Long J, Song C, Yan F, Zhou J, Zhou H, Yang B (2018). Non-TAL effectors from Xanthomonas oryzae pv. oryzae suppress peptidoglycan-triggered MAPK activation in rice. Front. Plant Sci. 9: 1857. doi: 10.3389/fpls.2018.01857

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

Mo X, Zhang L, Liu Y, Wang X, Bai J, Lu K, Zou S, Dong H, Chen L (2020). Three proteins (Hpa2, HrpF and XopN) are concomitant type III translocators in bacterial blight pathogen of rice. Front. Microbiol. 11: 1601. DOI: 10.3389/fmicb.2020.01601

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

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

Taylor KW, Kim JG, Su XB, Aakre CD, Roden JA, Adams CM, Mudgett MB (2012). Tomato TFT1 is required for PAMP-triggered immunity and mutations that prevent T3S effector XopN from binding to TFT1 attenuate Xanthomonas virulence. PLoS Pathog. 8: e1002768. DOI: 10.1371/journal.ppat.1002768

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