XopX

Author: Lucas Morinière & Sohini Deb
Internal reviewer: Coline Sciallano
Expert reviewer: Ramesh V. Sonti

Class: XopX
Family: XopX
Prototype: XopX (Xanthomonas euvesicatoria pv. euvesicatoria, ex Xanthomonas campestris pv. vesicatoria; strain 85-10)
RefSeq ID: WP_011346212.1 (699 aa)
3D structure: Unknown

XopX was discovered through the screening of a genomic cosmid library of X. euvesicatoria strain GM98-38 conjugated in X. campestris pv. campestris that allowed Xcc to elicit an Xcv cell death-like response when inoculated on N. benthamiana (Metz et al., 2005).

Translational fusions of XopX with the calmodulin-dependent adenylate cyclase domain of Bordetella pertussis (Cya) were exchanged by simple homologous recombination into the genomic copy of xopX of X. euvesicatoria strains 85* (constitutive hrp expression mutant), 85* ΔhrcV (T3SS-defective mutant) and wild-type GM98-38. All Cya fusion strains except 85* ΔhrcV still induced cell death response activity in N. benthamiana. Moreover, leaf extracts of N. benthamiana inoculated with these fusion strains were assayed for cAMP, and only strains with a functional T3SS showed an increase in cAMP levels due to translocation of the Cya reporter protein into the plant (Metz et al., 2005).

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 xopX, were significantly reduced in the Xanthomonas oryzae pv. oryzae ΔxrvC mutant compared with those in the wild-type strain PXO99^A (Liu et al., 2016).

• XopX was demonstrated to be cytotoxic when expressed in yeast, suggesting it may target a conserved eukaryotic cell process required for cell viability (Salomon et al., 2011).
• During infection of rice (Oryza sativa) with Xanthomonas oryzae pv. oryzae, XopX was shown to be an inhibitor of rice innate immune response, as it suppresses LipA-induced callose deposition (Sinha et al., 2013).
• XopX is required for the development of X. euvesicatoria- induced symptoms in the bacterial spot disease of tomato (Solanum lycopersicum) and pepper (Capsicum annuum). Indeed, it promotes ethylene production, and therefore chlorosis and plant cell death during infection by X. euvesicatoria of susceptible tomato and in transient expression assays in tobacco. Interestingly, it also suppresses flagellin-induced production of reactive oxygen species (ROS) while promoting the accumulation of pattern-triggered immunity (PTI) gene transcripts (Stork et al., 2015). Eventually, the complex behavior of XopX in planta, which combines activation and suppression of immunity-related plant responses at the same time, allows to classify this effector with the T3Es that activates the plant ‘default to death and defense’ response (Lindeberg et al., 2012; Stork et al., 2015).
• XopX is required for full virulence in Xanthomonas axonopodis pv. manihotisCIO151 strain. A Xam strain deleted for xopX showed decreased ability to produce symptoms in leaves of cassava. Also, the xopX KO reached more than one log unit lower populations than those observed for the wild‐type strain. This underlies the importance of this effector for disease developement (Medina et al., 2018).
• When expressed in Pseudomonas fluorescens 55 (Pf 55), a non‐pathogenic bacterium capable of eliciting PTI (callose deposit) in Arabidopsis Col‐0 plants, XopX is not able to reduce callose deposit, suggesting in these conditions, the effector is not able to suppress PTI. Using the same heterologuous system, XopX is not able to suppress the HR triggered by Pf 55 HopA1 on tobacco, suggesting that is these conditions, XopX do not act as an ETI suppressor. (Medina et al., 2018).
• When transiently expressed in N. benthamiana by Agrobacterium tumefaciens–mediated expression system, XopX from X. oryzae pv. oryzicola cause the nonhost HR at approximately 2 days (Li et al., 2015).
• A ∆xopX mutant strain of Xanthomonas phaseoli pv. manihotis (aka Xanthomonas axonopodis pv. manihotis) showed reduced growth in planta and delayed spread through the vasculature system of cassava (Mutka et al., 2016).
• Agrobacterium- mediated transient expression of both Xanthomonasoryzae pv.oryzaeXopQ 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 oryzaepv. oryzae 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).
• XopX(ACD57163) is one among the five classes of virulence genes found to be upregulated in theXanthomonas oryzaepv.oryzaeMAI1 strain during infection.xopXwas up-regulated at both 3 and 6 dai(Soto-Suárez et al., 2010).
• The xopX gene contributes to the virulence of Xanthomonas campestris pv. vesicatoria on hosts pepper and tomato. XopX targets the innate immune response, resulting in enhanced plant disease susceptibility (Metz et al., 2005).
• A Xanthomonas campestris pv.vesicatoriaxopX mutant strain could not induce cell death response inN. benthamiana, and could be complemented back to cell death response onN. benthamianain trans with plasmid subclones ofxopX(Metz et al., 2005).

Xanthomonas oryzae pv. oryzae XopX wild-type protein localizes mostly to the nucleus, but is also present to a lesser extent in the peripheral cytoplasm. However, the XopX S193A and XopX S477A mutants of XopX, that are defective in binding to the rice 14-3-3 proteins GF14d and GF14e, were found to be unable to localize to the nucleus, and were mostly observed in the cytoplasm (Deb et al., 2020).

Unknown.

• It has been suggested that XopX-triggering of plant cell death response was dependent on another cofactor delivered by the T3SS, yet still unknown (Metz et al., 2005).
• The Xanthomonas oryzae pv. oryzae XopX interacts with two of the eight rice 14-3-3 proteins, GF14d and GF14e. Mutants of XopX that are defective in 14-3-3 binding are also defective in suppression of immune responses, suggesting that interaction with 14-3-3 proteins is required for suppression of host innate immunity (Deb et al., 2020).
• Yeast two-hybrid, bimolecular fluorescence complementation (BiFC) and co-IP assays indicate that the Xanthomonas oryzae pv. oryzae XopX interacts with the type III effector XopQ (Debet al., 2020).

In xanthomonads

Yes, xopX homologs can be found in almost every sequenced Xanthomonas spp. strain, except X. albilineans and X. sacchari, making it an ancient Xanthomonas core T3E (Stork et al., 2015).

In other plant pathogens/symbionts

Related proteins (query cover > 80% and percent identity > 50 %) can be detected in several unclassified Burkholderiales (Xylophilus ampelinus, Rivibacter sp., Rhizobacter sp., Mitsuaria sp.) and in the Comamonadaceae (Hydrogenophaga taeniospiralis).

Deb S, Ghosh P, Patel HK, Sonti RV (2020). Interaction of the Xanthomonas effectors XopQ and XopX results in induction of rice immune responses. Plant J. 104: 332-350. DOI: 10.1111/tpj.14924

Li S, Wang Y, Wang S, Fang A, Wang J, Liu L, Zhang K, Mao Y, Sun W (2015). The type III effector AvrBs2 in Xanthomonas oryzae pv. oryzicola suppresses rice immunity and promotes disease development. Mol. Plant Microbe Interact. 28: 869-880. DOI: 10.1094/MPMI-10-14-0314-R Lindeberg M, Cunnac S, Collmer A (2012). Pseudomonassyringae type III effector repertoires: last words in endless arguments. Trends Microbiol. 20: 199-208. DOI: 10.1016/j.tim.2012.01.003

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

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

Metz M, Dahlbeck D, Morales CQ, Sady BA, Clark ET, Staskawicz BJ (2005). The conserved Xanthomonas campestris pv. vesicatoria effector protein XopX is a virulence factor and suppresses host defense in Nicotiana benthamiana. Plant J. 41: 801-814. DOI: 10.1111/j.1365-313X.2005.02338.x

Mutka AM, Fentress SJ, Sher JW, Berry JC, Pretz C, Nusinow DA, Bart R (2016). Quantitative, image-based phenotyping methods provide insight into spatial and temporal dimensions of plant disease. Plant Physiol. 172: 650-660. DOI: 10.1104/pp.16.00984

Salomon D, Dar D, Sreeramulu S, Sessa G (2011). Expression of Xanthomonas campestris 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

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

Soto-Suárez M, Bernal D, González C, Szurek B, Guyot R, Tohme J, Verdier V. In planta gene expression analysis of Xanthomonas oryzae pathovar oryzae, African strain MAI1. BMC Microbiol. 2010 Jun 11;10:170. DOI: 10.1186/1471-2180-10-170.

Stork W, Kim JG, Mudgett MB (2015). Functional analysis of plant defense suppression and activation by the Xanthomonas core type III effector XopX. Mol. Plant. Microbe Interact. 28: 180-194. DOI: 10.1094/MPMI-09-14-0263-R