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Molecular Diagnosis and Diversity for Regulated Xanthomonas
Bacterial virulence factors
This DokuWiki is based upon work from COST Action CA16107 EuroXanth, supported by COST (European Cooperation in Science and Technology)
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Author: Katarina Gašić
Internal reviewer: Daiva Burokienė
Expert reviewer: 
Class: XopJ
Family: XopJ4
Prototype: AvrXv4 (Xanthomonas euvesicatoria pv. perforans, ex Xanthomonas campestris pv. vesicatoria; race T3 strain 91-118)
RefSeq ID: WP_008572727.1 (359 aa)
Synonym: AvrXv4
3D structure: Unknown
How discovered?
An avirulence gene named avrXv4 was isolated by mobilizing a total of 600 clones from a genomic DNA library of the T3 strain 91-118 into the X. campestris pv. vesicatoria strain ME90, virulent on L. pennellii. One cosmid clone, pXcvT3-60 (29-kb insert), induced HR in resistant plants. The avirulent phenotype of pXcvT3-60 was confirmed by comparing growth rates in planta and electrolyte leakages among transconjugants carrying a mutated or intact clone with the wild-type T3 strain 91-118. A 1.9-kb DNA fragment contained within a 6.8-kb active subclone was sequenced and was determined to carry an open reading frame of 1,077 bp (Astua-Monge et al., 2000).
To ascertain if X. campestris pv. vesicatoria (Xcv) translocates AvrXv4 via the hrp-encoded TTSS into plant cells during infection, the calmodulin-dependent adenylate cyclase domain (Cya) of Bordetella pertussis cyclolysin as a sensitive reporter protein was employed. It was constructed a translational C-terminal fusion of the Cya reporter to the mature AvrXv4 protein (359 amino acids [aa]) (AvrXv4-Cya) and to the first 100 aa residues of AvrXv4 (AvrXv41-100-Cya). Cya enzymatic activity requires eukaryotic calmodulin for the production of cAMP. Therefore, the Cya reporter protein produced in the bacterium must be translocated into the eukaryotic cell to be activated (Roden et al., 2004). Results of experiments showed that Xcv secretes AvrXv4 via the Hrp TTSS and translocates it directly through the cell wall into the interior of the plant cell. Furthermore, the N-terminal 100 aa of AvrXv4 are sufficient to target the type III-dependent secretion and translocation of the Cya reporter to plant cells (Roden et al., 2004).
The immune pathway reasponsible for the perception of the Xanthomonas perforans effector XopJ4 wa sidentified in the plant Nicotiana benthamiana. Transient complemmentation assays were performed to determine the functionality of xopJ4 gene variants and coimmunoprecipitation assays were used to gain insight into molecular mechanism of the pathway (Schultink et al., 2019).
Presence of a PIP box (TTCGC-N15-TTCGC) (Astua-Monge et al., 2000).
AvrXv4 is an avirulence protein in X. campestris pv. vesicatoria strain 91-118 that induces an XV-dependent HR in the wild tomato relative Lycopersicon pennellii (Astua-Monge et al., 2000; Roden et al., 2004; Sharlach et al., 2013). AvrXv4-dependent HR also is elicited in Nicotiana benthamiana plants when they are infected with Xanthomonas strains expressing untagged AvrXv4 and HA-tagged AvrXv4-HA (Roden et al., 2004). AvrXv4 recognition by resistant plants requires a functional protease catalytic core, the domain that is conserved in all of the putative YopJ-like cysteine proteases. AvrXv4 expression in planta leads to a reduction in SUMO-modified proteins, demonstrating that AvrXv4 possesses SUMO isopeptidase activity. YopJ-like effector AvrXv4 encodes a type III SUMO protease effector that is active in the cytoplasmic compartment of plant cells (Roden et al., 2004).
N. benthamiana ethyl methanesulfonate (EMS) mutants deficient for XopJ4 perception were idetified as having loss of function mutations in the gene encoding the nucleotide binding, leucine rich repeat (NLR) protein NbZAR1. Silecing of a recertor-like cytoplasmic kinase family XII gene, subsequently named XOPJ4 IMMUNITY 2 (JIM2), blocks perception of XopJ4 (Schultink et al., 2019).
Localization
AvrXv4 is localized to the plant cytoplasm (Roden et al., 2004).
The xopJ4 gene was located between phage associated genes (Timilsina et al., 2019).
Because in planta expression of AvrXv4 from X. campestris pv. vesicatoria leads to a reduction of SUMO-conjugated plant proteins, AvrXv4 presumably acts as a SUMO protease on yet unknown plant target proteins (Roden et al., 2004).
In xanthomonads
Yes (Xanthomonas perforans) (Astua-Monge et al., 2000; Roden et al., 2004; Timilsina et al., 2016).
In other plant pathogens/symbionts
Yes (Ralstonia solanacearum) (Lavie et al., 2002).
The predicted AvrXv4 protein exhibits high similarity to members of family of bacterial proteins comprising AvrRxv, AvrBsT, AvrA, YL4O (Astua-Monge et al., 2000).
Astua-Monge G, Minsavage GV, Stall RE, Vallejos CE, Davis MJ, Jones JB (2000). Xv4-avrXv4: a new gene-for-gene interaction identified between Xanthomonas campestris pv. vesicatoriarace T3 and wild tomato relative Lycopersicon pennellii. Mol. Plant Microbe Interact. 13: 1346-1355. DOI: 10.1094/MPMI.2000.13.12.1346
Lavie M, Shillington E, Eguiluz C, Grimsley N, Boucher C (2002). PopP1, a new member of the YopJ/AvrRxv family of type III effector proteins, acts as a host-specificity factor and modulates aggressiveness of Ralstonia solanacearum. Mol. Plant Microbe Interact. 15: 1058-1068. DOI: 10.1094/MPMI.2002.15.10.1058
Roden J, Eardley L, Hotson A, Cao Y, Mudgett MB (2004). Characterization of the Xanthomonas AvrXv4 effector, a SUMO protease translocated into plant cells. Mol. Plant Microbe. Interact. 17: 633-643. DOI: 10.1094/MPMI.2004.17.6.633
Sharlach M, DahlbeckD, LiuL, ChiuJ, Jiménez-GómezJM, KimuraS, KoenigD, MaloofJN, SinhaN, MinsavageGV, JonesJB, StallRE, StaskawiczBJ (2013). Fine genetic mapping of RXopJ4, a bacterial spot disease resistance locus from Solanum pennellii LA716. Theor Appl Genet. 126: 601-609. DOI: 10.1007/s00122-012-2004-6
Schultink A, Qi T, Bally J, Staskawicz B (2019). Using forward genetics in Nicotiana benthamiana to uncover the immune signaling pathway mediating recognition of the Xanthomonas perforans effector XopJ4. New Phytologist 221: 1001-1009. DOI: 10.1111/nph.15411
Timilsina S, Abrahamian P, Potnis N, Minsavage GV, White FF, Staskawicz BJ, Jones JB, Vallad GE, Goss EM (2016). Analysis of sequenced genomes of Xanthomonas perforans identifies candidate targets for resistance breeding in tomato. Phytopathology 106: 1097-1104. DOI: 10.1094/PHYTO-03-16-0119-FI
Timilsina S, Pereira-Martin JA, Minsavage GV, Iruegas-Bocardo F, Abrahamian, Potnis N, Kolaczkowski B, Vallad GE, Goss EM, Jones JB (2019). Multiple recombination events drive the current genetic structure of Xanthomonas perforans in Florida. Front Microbiol. 10: 448. DOI: 10.3389/fmicb.2019.00448
