Author: Marlène Lachaux
Internal reviewer: Joël F. Pothier
Expert reviewer:

Class: XopZ
Family: XopZ
Prototype: XopZ (Xanthomonas oryzae pv. oryzae; strain PXO99^A )
RefSeq ID: ACD59124.1 and ACD59315.1 (=PXO_01041 and PXO_06152, respectively as PXO99^A contains two identical copies of the gene due to a 212 kb duplication in the genome (Song et al., 2010). These GenBank entries are only 1,371 aa long whereas the initial description (Song and Yang, 2010) mentions 1,414 aa. AJQ87647 1,411 aa in Xanthomonas oryzae pv. oryzicola CFBP 7342 might be preferred).
3D structure: Unknown. The N-terminus of XopZ_PXO99, contains two Nuclear Localization Signals (NLS) signals and several Nuclear Export Signals (NES) (Zhou et al., 2015).

The first mention of XopZ as an homolog of HopAS1 in Xanthomonas oryzae MAFF311018 was made by Furutani et al. (2009). Indeed, the locustag XOO2402 (BAE69157; 1,288 aa) was shown to share homology with known Hrp outer proteins (Hops) of Pseudomonas syringae strains (Lindeberg et al., 2005).

In 2009, the generation of mutants for 18 non-TAL type 3 effector genes in Xoo strain PXO99^A allowed to investigate the function of several T3Es. Among them XopZ (PXO_06152 and PXO_01041) was reported to contribute to the full virulence of the strain PXO99^A (Ryan et al., 2009; Song and Yang, 2010).

XopZ2 was described in Potnis et al., 2011 as a novel candidate effector gene upstream of hrpW in Xanthomonas vesicatoria strain 1111 (=ATCC 35937) (EGD08510.1=XVE_3221) and Xanthomonas gardneri strain 101 (=ATCC 19865) (EGD18683.1=XGA_2762; Potnis et al., 2011). It was also shown to be functional i.e. as being translocated using a reporter gene assay (AvrBs2-based assay; Potnis et al., 2011). The pairwise sequence identity below 50% warrants assigning these two proteins to a new family within the xopZ class, named xopZ2 (Potnis et al., 2011)

The secretion of XopZ in planta was shown using a B. pertussis Cya translocation reporter assay (Furutani et al., 2009). With a PIP box 58 bp upstream of the predicted translation start site, xopZ_PXO99 gene is certainly inducible in planta and regulated through the hypersensitive reaction and pathogenicity (hrp) regulatory network (Song and Yang, 2010). PXO99^A and an hrpG mutant were grown in Nutrient Broth (NB) or Xanthomonas hrp-inducing medium (XOM2) (Song and Yang, 2010). The expression of xopZ_PXO99 was only observed, by RT-PCR, in XOM2 medium and was hrpG dependent (Song and Yang, 2010).

The xopZ gene was shown to be expressed in a hrpG-dependent manner. A PIP box (TTCTC-N₁₅-TTCGC) was identified 58 bp upstream of the predicted translation start site (Song and Yang, 2010).

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 xopZ, 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).

PXO99^A contains two identical copies of the gene due to a duplication of 212 kb in the genome. However, a deletion of one xopZ gene did not affect pathogenicity or bacterial growth in plants, while strains with mutations in both copies of xopZ_PXO99 displayed reduced virulence in terms of lesion length and bacterial multiplication compared with the wild type strain PXO99^A . The introduction of one genomic copy of xopZ_PXO99 restores the mutant to full virulence. To test whether XopZ_PXO99 inhibits the host cell-wall-associated defense responses (PTI), leaves of Nicotiana benthamiana were infiltrated with Agrobacterium cells with and without xopZ_PXO99 under the control of the cauliflower mosaic virus 35S promoter 24 hours preceding inoculation of the same leaves with a T3SS mutant of PXO99^A (ME7). Twenty-four hours after inoculation, leaves inoculated with ME7 had more callose depositions than the leaves inoculated with Agrobacterium spp. expressing xopZ_PXO99 . This results suggesting a role for XopZ_PXO99 in interfering with host innate immunity (PTI) during X. oryzae pv. oryzae infection (Song et al., 2010). Besides, Western blot analysis with p44/42 MAP kinase antibody clearly showed that XopN, XopV and XopZ inhibited the peptidoglycan(PNG)-induced phosphorylation of OsMAPKs. Expression of all Xop effectors were verified by immunoblotting with anti-HA antibody. Thus, expression of three Xop effectors from PXO99^A in rice protoplasts results in compromised OsMAPK activation induced by PGN, highlighting their putative virulence functions during pathogenesis (Long et al., 2018).

A role of XopZ in full virulence was also clearly shown in Xanthomonas axonopodis pv. manihotis CIO151 but not in PTI or ETI supression, at least under the tested conditions, as on the contrary to XopZ of X. oryzae pv. oryzae PXO99, no reduction of callose deposition was observed (Medina et al., 2017).

XopZ_PXO99 localizes in the cytoplasm and nucleus of the plant cell (Zhou et al., 2015).

XopZ_PXO99 functions as a suppressor of LipA-induced innate immune responses since the mutation of XopZ partially compromises virulence while quadruple mutant of xopN/xopQ/xopX/xopZ induces calloses deposition just similarly to Xoo T3SS-mutant in rice leaves (Sinha et al., 2013). The function of XopZ is also to stabilize a putative host E3 ubiquitin ligase protein PBP (s-ribonuclease) in the nucleus and prevents its degradation-mediated by a cysteine protease (C1A) in plant cells. XopZ may function to interfere with the homeostatic state of the negative regulator (PBP) in immune system in rice, and subvert the plant immune response (Zhou et al., 2015).

XopZ interacts with a putative host E3 ubiquitin ligase protein PBP (s-ribonuclease) in vitro and in vivo. Regions containing 193 aa - 225 aa of PBP is required for interacting with XopZ. PBP is a negative regulator of host immune response based on the disease phenotype in PBP-knockout rice plants. C1A directly interacts and strongly degrades PBP through its cysteine protease activity, leading to a homeostatic state of PBP in plant cells (Zhou et al., 2015).

Yes, found to be conserved in all Xanthomonas spp. (whose genomes have been sequenced) with the exception of some clade-1 strains (e.g. X. albilineans) (Song and Yang, 2010; Sinha et al., 2013).

Related genes are also found in several Pseudomonas syringae pathovars (HopAs1 relatives), a few strains of Ralstonia solanacearum (AWR proteins), and the AAC00-1 strain of Acidovorax avenae subsp. citrulli (Song and Yang, 2010).

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

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

Lindeberg M, Stavrinides J, Chang JH, Alfano JR, Collmer A, Dangl JL, Greenberg JT, Mansfield JW, Guttman DS (2005). Proposed guidelines for a unified nomenclature and phylogenetic analysis of type III Hop effector proteins in the plant pathogen Pseudomonas syringae. Mol Plant Microbe Interact. 18: 275-282. DOI: 10.1094/MPMI-18-0275

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

Potnis N, Krasileva K, Chow V, Almeida NF, Patil PB, Ryan RP, Sharlach M, Behlau F, Dow JM, Momol M, White FF, Preston JF, Vinatzer BA, Koebnik R, Setubal JC, Norman DJ, Staskawicz BJ, Jones JB (2011). Comparative genomics reveals diversity among xanthomonads infecting tomato and pepper. BMC Genomics 12: 146. DOI: 10.1186/1471-2164-12-146

Ryan RP, Koebnik R, Szurek B, Boureau T, Bernal A, Bogdanove A, Dow JM (2009). Passing GO (gene ontology) in plant pathogen biology: a report from the Xanthomonas Genomics Conference. Cell. Microbiol. 11: 1689-1696. DOI: 10.1111/j.1462-5822.2009.01387.x

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

Song C, Yang B (2010). Mutagenesis of 18 type III effectors reveals virulence function of XopZ _PXO99 in Xanthomonas oryzae pv. oryzae. Mol. Plant Microbe Interact. 23: 893-902. DOI: 10.1094/MPMI-23-7-0893

Zhou J (2015). Host target genes of the Xanthomonas oryzae pv. oryzae type III effectors for bacterial blight in rice. Doctoral Thesis, Iowa State University, USA. PDF: lib.dr.iastate.edu/etd/14469/