The Type III Effector XopS from //Xanthomonas//

Author: Gábor Rákheli
Internal reviewer: Fernando Tavares
Expert reviewer: Sujan Timilsina

Class: XopS
Family: XopS
Prototype: XCV0324 (Xanthomonas euvesicatoria pv. euvesicatoria, ex Xanthomonas campestris pv. vesicatoria; strain 85-10)
GenBank ID: CAJ21955.1 (308 aa, perhaps 5 aa too long)
RefSeq ID: WP_039418869.1 (292 aa, perhaps 11 aa too short)
3D structure: Unknown

xopS was putatively identified by the presence of a plant-induced promoter (PIP) box, a lower G+C content suggesting acquisition by HGT, and co-expression with other T3E genes (Schulze et al., 2012). Deficient mutants and overexpression studies revealed that XopB and XopS contribute to disease symptoms and bacterial growth, and suppress plant defense gene expression (Schulze et al., 2012).

Type III secretion (T3S) assays of XopS-AvrBs3Δ2 fusion proteins indicated that XopS is secreted and translocated into the plant cells through T3S, inducing HR in Bs3 resistant pepper leaves. Further supporting the identify of XopS as a T3E, it was shown that deletion mutants (ΔxopS) cause a considerable attenuation of disease symptoms in pepper (Schulze et al., 2012).

For expression in X. campestris pv. vesicatoria (Xcv), xopS was amplified from strain 85-10 and cloned into the Golden Gate‐compatible expression vector pBRM (Schulze et al., 2012). To generate avrBs3Δ2 fusions, the promoters and 5′ coding sequences of xopS was amplified by PCR from genomic DNA of Xcv 85-10, cloned into pENTR/D‐TOPO and recombined into pL6GW356 (Schulze et al., 2012). To generate deletions of xopS, 0.6–1kb fragments upstream and downstream of the respective gene were amplified from genomic DNA of Xcv 85-10 by PCR using oligonucleotides harboring appropriate restriction sites. Fragments were cloned into the suicide vectors pOK1 (Schulze et al., 2012).

HrpG- and HrpX-dependent co-regulation with the T3S system (Schulze et al., 2012). Presence of a PIP and -10 box (TTCGB‐N₁₅ ‐TTCGB‐N_30–32 ‐YANNNT) (Schulze et al., 2012).

To study the contribution of the T3Es to bacterial virulence, the effector gene was individually deleted in Xcv strain 85‐10, and the mutant was inoculated into leaves of susceptible ECW pepper plants. In addition, induction of the HR in pepper ECW‐10R was analyzed, which is based on the recognition of the T3E AvrBs1 by the Bs1 resistance gene (Schulze et al., 2012). Schulze et al. 2012 studied XopS along with XopB in their study. Deletion of xopB or xopS led to significantly reduced disease symptoms, whereas the HR induction was not impaired. The mutant phenotypes of 85-10ΔxopB and 85-10ΔxopS were complemented by ectopic expression of the respective effector gene, suggesting that reduced virulence was not caused by polar effects of the deletions on downstream genes. Although the growth of both individual effector mutants in ECW plants did not differ significantly from that of the wild‐type strain), multiplication of an 85‐10ΔxopBΔxopS double mutant was reduced significantly, suggesting that XopB and XopS fulfill redundant functions. To identify additional virulence phenotypes, as well as defense reactions, mediated by the analyzed T3Es, leaves of pepper ECW, N. benthamiana and N. tabacum were inoculated with Agrobacterium strains mediating the in planta expression of the effector genes fused to GFP. These experiments confirmed that XopS (similar to XopB, XopG, XopM) trigger cell death in different Solanaceae (Schulze et al., 2012). XopS is involved in the severity of disease symptoms, the promotion of bacterial growth and the suppression of PTI (Schulze et al., 2012).

XopS_Xcv85-10 inhibited proteasomal degradation of WRKY40, a transcriptional regulator of defense gene expression. Virus-induced gene silencing of WRKY40 in pepper enhanced plant tolerance to Xcv infection, indicating that WRKY40 represses immunity. Stabilization of WRKY40 by XopS reduced the expression of its targets, which included salicylic acid-responsive genes and the jasmonic acid signaling repressor JAZ8. Xcv bacteria lacking XopS displayed significantly reduced virulence when surface inoculated onto susceptible pepper leaves. XopS delivery by Xcv, as well as ectopic expression of XopS in Arabidopsis thaliana or Nicotiana benthamiana, prevented stomatal closure in response to bacteria and biotic elicitors. Silencing WRKY40 in pepper or N. benthamiana abolished XopS’s ability to prevent stomatal closure. These findings suggests that XopS interferes with both preinvasion and apoplastic defense by manipulating WRKY40 stability and downstream gene expression, eventually altering phytohormone crosstalk to promote pathogen proliferation (Raffeiner et al., 2022).

Unknown.

Unknown.

XopS_Xcv85-10 interacts with WRKY40, a transcriptional regulator of defense gene expression (Raffeiner et al., 2022).

Yes (e.g., Xanthomonas euvesicatoria, X. perforans, X. citri) (Barak et al., 2016; Fonseca et al., 2019).

Unknown

Barak JD, Vancheva T, Lefeuvre P, Jones JB, Timilsina S, Minsavage GV, Vallad GE, Koebnik R (2016). Whole-genome sequences of Xanthomonas euvesicatoria strains clarify taxonomy and reveal a stepwise erosion of type 3 effectors. Front. Plant Sci. 7: 1805. DOI: 10.3389/fpls.2016.01805

Fonseca NP, Patané JSL, Varani AM, Felestrino EB, Caneschi WL, Sanchez AB, Cordeiro IF, Lemes CGC, Assis RAB, Garcia CCM, Belasque Jr. J, Martins Jr J, Facincani AP, Ferreira RM, Jaciani FJ, Almeida NF, Ferro JA, Moreira LM, Setubal JC (2019). Analyses of seven new genomes of Xanthomonas citri pv. aurantifolii strains, causative agents of citrus canker B and C, show a reduced repertoire of pathogenicity genes. Front Microbiol. 10: 2361. DOI: 10.3389/fmicb.2019.02361

Raffeiner M, Üstün S, Guerra T, Spinti D, Fitzner M, Sonnewald S, Baldermann S, Börnke F (2022). The Xanthomonas type-III effector XopS stabilizes CaWRKY40a to regulate defense responses and stomatal immunity in pepper (Capsicum annuum). Plant Cell 34: 1684-1708. DOI: 10.1093/plcell/koac032

Schulze S, Kay S, Büttner D, Egler M, Eschen-Lippold L, Hause G, Krüger A, Lee J, Müller O, Scheel D, Szczesny R, Thieme F, Bonas U (2012). Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity. New Phytol. 195: 894-911. DOI: 10.1111/j.1469-8137.2012.04210.x

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