- Short genome report
- Open Access
Draft genome sequence for virulent and avirulent strains of Xanthomonas arboricola isolated from Prunus spp. in Spain
Standards in Genomic Sciencesvolume 11, Article number: 12 (2016)
Xanthomonas arboricola is a species in genus Xanthomonas which is mainly comprised of plant pathogens. Among the members of this taxon, X. arboricola pv. pruni, the causal agent of bacterial spot disease of stone fruits and almond, is distributed worldwide although it is considered a quarantine pathogen in the European Union. Herein, we report the draft genome sequence, the classification, the annotation and the sequence analyses of a virulent strain, IVIA 2626.1, and an avirulent strain, CITA 44, of X. arboricola associated with Prunus spp. The draft genome sequence of IVIA 2626.1 consists of 5,027,671 bp, 4,720 protein coding genes and 50 RNA encoding genes. The draft genome sequence of strain CITA 44 consists of 4,760,482 bp, 4,250 protein coding genes and 56 RNA coding genes. Initial comparative analyses reveals differences in the presence of structural and regulatory components of the type IV pilus, the type III secretion system, the type III effectors as well as variations in the number of the type IV secretion systems. The genome sequence data for these strains will facilitate the development of molecular diagnostics protocols that differentiate virulent and avirulent strains. In addition, comparative genome analysis will provide insights into the plant-pathogen interaction during the bacterial spot disease process.
Xanthomonas arboricola  are plant associated bacteria in nine pathovars with a diverse range of biotic relationships [2, 3]. Within this taxon, plant pathogenic strains with non-pathogenic strains have been described. Bacterial spot of Prunus spp. ( X. arboricola pv. pruni), bacterial blight of Juglans spp. ( X. arboricola pv. juglandis) and Corylus spp. ( X. arboricola pv. corylina) are among the most harmful diseases of these tree hosts. These bacterial diseases are distributed worldwide and the causal bacteria are regulated in several countries including the European Union, where X. arboricola pv. pruni is a quarantine pathogen [4, 5].
Within the pathovars, X. arboricola pv. pruni is a major threat to cultivated, exotic and ornamental Prunus species. This bacterium has been identified as a pathogen of P. armeniaca , P. avium , P. buergeriana , P. cerasus P. crassipes , P. davidiana , P. domestica , P . donarium, P. dulcis , P. laurocesasus , P. mume , P. persica and P. salicina . During the last decade, some local outbreaks of bacterial spot in Spain have been reported on almond, peach, nectarine and plum . For initial characterization of the bacterial strains isolated from Spanish outbreaks of bacterial spot, we performed a polyphasic study based on a multilocus sequence analysis, as well as some phenotypic characters . After the characterization that showed the presence of different molecular and phenotypic variants, selected strains were analysed to assess the differences at the whole genome level.
Genome sequencing of X. arboricola strains has been completed for five strains isolated from walnut, three from peach, two from Musa sp., one from almond , one from barley  and one from Turkish hazel . Genome sequencing includes the plasmid pXap41 , present in the X. arboricola pv. pruni strains. All these sequences have been deposited in the NCBI database. Four genome sequences are available for pathogenic strains from Prunus , identified as X. arboricola pv. pruni. However, with the exception of the strain CITA 33 isolated from almond ( P. amygdalus , syn. P. dulcis ) in Spain , no detailed information about features of those genomes have been published. In the same way, there are no sequenced strains isolated from Japanese plum ( P. salicina ) or cherry rootstock ( P. mahaleb ). In addition, no avirulent strain of X. arboricola from Prunus spp. has been analysed at the whole-genome level. The occurrence of avirulent strains is of particular importance for a quarantine pathogen like X. arboricola pv. pruni with respect to accurate diagnosis of virulent strains.
Herein we present draft genome sequences for two X. arboricola strains: an avirulent strain, CITA 44, isolated from P. mahaleb, and X. arboricola pv. pruni strain, IVIA 2626.1, isolated from P. salicina cv. Fortuna, which differs from other sequenced strains in phenotypical features and virulence on several hosts . The genome analysis of these two strains as well as comparison with other related strains should provide insight into the genetics of the pathogenesis process in X. arboricola strains associated with the bacterial spot disease of stone fruits and almond.
Classification and features
Strain CITA 44 was isolated in 2009 from asymptomatic leaves of Santa Lucía SL-64 cherry rootstock ( P. mahaleb ) in a nursery located in the north-eastern Spanish region of Aragón. This strain showed flagella associated swarming and swimming motility on 0.5 % agar PYM plates and 0.3 % agar MMA plates, respectively. Additionally, strain CITA 44 showed type IV pili associated twitching motility in the interstitial surface between 1 % agar PYM layer and the plastic plate surface. According to the atomized oil assay , this strain produced surfactant compounds on 1.5 % agar LB plates after 24 h at 27 °C. In accordance with a detached leaf assay, conducted with a cotton swap damped with 1 × 108 CFU/ml, on almond cv. Ferraduel, apricot cv. Canino, peach cv. Calanda and European plum ( P. domestica ) cv. Golden Japan, X. arboricola strain CITA 44 did not cause bacterial spot symptoms at 28 days post inoculation (dpi). Despite this lack of symptoms, the bacterium could be re-isolated after such period.
X. arboricola pv. pruni strain IVIA 2626.1 was isolated from symptomatic leaves of Japanese plum ( P. salicina cv. Fortune) in the southwestern Spanish region of Extremadura in 2002. This strain showed swarming, swimming and twitching type motility as well as production of surfactant compounds in the same culture conditions described above for strain CITA 44. In addition, according to the detached leaf assay described previously, strain IVIA 2626.1 was able to produce bacterial spot symptoms on almond, peach and European plum but not on apricot after 28 dpi.
Classification of the strains was performed using an MLSA approach based on the partial sequences of the housekeeping genes atpD, dnaK, efP, fyuA, glnA, gyrB and rpoD of the strains CITA 44 and IVIA 2626.1 as well as related strains of X. arboricola . Nucleotide sequences were aligned with Clustal W and both ends of each alignment were trimmed (atpD 750 bp, dnaK 759 bp, efP 339 bp, fyuA 753 bp, glnA 675 bp, gyrB 735 bp and rpoD 756 bp) and concatenated to a total length sequence of 4,620 nucleotide positions. The phylogenetic tree was constructed using the maximum likelihood method implemented in MEGA 6.0  using 1,000 bootstrap re-samplings. According to the phylogenetic analysis, strain CITA 44 belongs to the species X. arboricola , nevertheless, this strain could not be associated to any of the pathovars of this species. The concatenated sequence similarity among this strain and the other X. arboricola strains analysed varied from 97.08 % to 98.79 %. In contrast, strain IVIA 2626.1 was clustered in a group with the pathotype strain X. arboricola pv. pruni CFBP 2535, isolated from P. salicina in New Zealand, with a sequence similarity of 100 %.
X. arboricola CITA 44 and X. arboricola pv. pruni IVIA 2626.1 strains are Gram-negative, non-sporulating, rod-shaped, motile cells with a single polar flagellum. Rod-shaped cells of CITA 44 are approximately 0.6 μm in width and 1.4–2.5 μm in length. Rod-shaped cells of IVIA 2626.1 are approximately 0.7 μm in width and 1.7–2.5 μm in length. These strains formed 2.0–3.0 mm colonies within 48 h at 27 °C on YPGA 1.5 % agar plates . Both strains formed mucoid, circular, yellow colonies with a convex elevation and an entire margin (Fig. 1). Strains CITA 44 and IVIA 2626.1 grew in the nutritive culture media PYM  and LB , as well as in the minimal medium A . According to the Biolog GN2 system, both strains metabolized α-D-glucose, α-keto glutamic acid, bromosuccinic acid, D-cellobiose, D-fructuose, D-mannose, D-psicose, D-threalose, glycyl-L-glutamic acid, L-glutamic acid, L-serine, pyruvic acid methyl ester, succinic acid, succinic acid mono-methyl-ester, sucrose and Tween 40. The carbon compound D-saccharic acid was only utilized by strain CITA 44. Dextrin and L-proline were only metabolized by strain IVIA 2626.1. In addition to this analysis, strain CITA 44 hydrolysed casein and starch, while strain IVIA 2626.1 did not (Table 1).
Minimum information about genome sequence  of X. arboricola strain CITA 44 and X. arboricola pv. pruni strain IVIA 2626.1, as well as their phylogenetic position, are provided in Table 1 and Fig. 2.
Genome sequencing information
Genome project history
X. arboricola strain CITA 44 and X. arboricola pv. pruni strain IVIA 2626.1 were selected for comparative whole sequencing analysis as X. arboricola strains isolated from Prunus spp. with several different phenotypic characters including virulence. Comparative genomics among the avirulent strain CITA 44 and the available Prunus-pathogenic strains including IVIA 2626.1 should be useful for identifying the molecular determinants associated with pathogenesis as well as those associated with host resistance and for diagnostic characterization of X. arboricola strains causing bacterial spot of Prunus spp. Whole Genome Shotgun Projects have been deposited at DDBJ/EMBL/GenBank under the accession numbers LJGM00000000 and LJGN00000000. The versions described in this paper are versions LJGM01000000 and LJGN01000000. Table 2 summarizes the project information and its association with MIGS.
Growth conditions and genomic DNA preparation
X. arboricola strain CITA 44 and X. arboricola pv. pruni strain IVIA 2626.1 are deposited and available at the bacterial collections of the Instituto Valenciano de Investigaciones Agrarias (IVIA, Valencia, Spain) and the Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITA, Zaragoza, Spain). Both strains were streaked on 1.5 % agar LB plates and were grown for 48 h at 27 °C. A single colony of each strain was inoculated separately in 30 ml of LB broth and grown on an orbital shaker for 24 h at 27 °C. DNA from pure bacterial cultures was extracted using a QIAamp DNA miniKit (Qiagen, Barcelona, Spain) according to the manufacturer instructions. DNA quality and quantity were determined by 1 % agarose gel electrophoresis, as well as using the Qubit flurometer (Invitrogen) according to the Quant-it dsDNA BR Assay Kit (Invitrogen) manufacturer instructions, and by a spectrophotometry (NanoDrop 2000 spectrophotometer, Thermo Scientific). A 2.0 μg/μl aliquot of 200 ng/μl sample was submitted for the sequencing.
Genome sequencing and assembly
The draft genome sequences for strains CITA 44 and IVIA 2626.1 were generated at the STAB VIDA Next Generation Sequencing Laboratory (Caparica, Portugal) using the Ion Torrent sequencing technology. Draft genome assembly of strain CITA 44 was based on 3,060,638 usable reads with a total base number of 948,933,067. The mean read length was 361.70 ± 93.50 and the mode read length was 385 bp. The draft genome assembly of IVIA 2626.1 was based on 2,317,319 reads, with a total base number of 461,361,072. The mean read length and the mode read length for this strain were 201.80 ± 85.30 bp and 241 bp, respectively. Genomic assemblies were constructed using MIRA 4.0 . From the total of contigs generated, only those with a contig size above 500 bp and an average coverage above 99 in the case of CITA 44, and 40, in the case of IVIA 2626.1 were considered significant. Finally, 71 contigs (N50 = 120,981 bp; largest contig = 352,479 bp; average coverage = 198X) were generated for strain CITA 44 and for strain IVIA 2626.1, 214 contigs (N50 = 47,650; largest contig = 115,385; average coverage = 92X) were generated.
The assembled draft genome for both strains was annotated using the RAST platform and the gene-caller GLIMMER 3.02 [21, 22]. RNAmmer version 1.2  and tRNAscan-SE version 1.21  were used to predict rRNAS and tRNAS, respectively. Signal peptides and transmembrane domains were determined using the SignalP 4.1 server  and the TMHMM server version 2.0 , respectively. Assignment of genes to the COG database  and Pfam domains  was performed with the NCBI conserved domain database using an expected value threshold of 0.001 .
Major structural components associated with the flagellum [30, 31], the type IV pilus , the type III secretory system [33, 34] and the type III effectors [35, 36], as well as the type IV secretory system and effectors [37–39], were identified in the draft genome sequence for each strain. Initially, the query of those genes was based on the coding sequence regions automatically annotated by RAST, and were confirmed using the BLASTn and BLASTx tools available at NCBI. Those components which were not automatically annotated were found in the genome sequence using the progressive Mauve alignment method . Nucleotide sequences of the genes used for these alignments were obtained from other xanthomonads in the NCBI gene database. Finally, the nucleotide sequence of the aligned regions was analysed using the BLAST approaches mentioned above. Those sequences with query coverage and identity percentage higher than 90 % were annotated. Additionally, the core components of the T3SS and T4SS were searched using the T346Hunter application . T3Es and T4Es genes were predicted using the Effective database  after selection of the “gram-” parameter as organism type and the “plant set” parameter as classification module, and the SecReT4 tool , respectively. All the predicted genes were corroborated and annotated according to the BLAST parameters mentioned above.
The draft genome sequence of X. arboricola strain CITA 44 was 4,760,482 bp in length with an average GC content of 65.8 %, which is similar to that for other genomes of this species (65.4 to 66.0 %) reported in the NCBI genome database. For this strain, 4,306 genes were predicted and 4,250 were determined as protein coding genes. From these protein coding genes, 3,330 genes were assigned to a putative function and the remaining 920 were designated as hypothetical proteins. This strain presented 3 rRNA and 53 tRNA genes. In the case of the X. arboricola pv. pruni strain IVIA 2626.1, the draft genome sequence was 5,027,671 bp in length with an average GC content of 65.4 %, which is the same as for other strains of X. arboricola pv. pruni according to the NCBI database. A total of 4,770 genes were predicted and, among them, 4,720 were predicted as protein coding genes with 69.17 % assigned to a function and 30.83 % designated as hypothetical proteins. 50 RNA genes (3 rRNA and 47 tRNA genes) were predicted for this strain. The properties and characteristics associated with these genomes are presented in Table 3. The classification of the predicted protein coding genes into COG functional categories  is summarized in Fig. 3 and Table 4.
Insights from the genome sequence
Based on the phenotypic differences between CITA 44 and IVIA 2626.1 strains, selected genes associated with motility and pathogenicity were analysed (Table 5). No differences were observed for the structural components associated with bacterial flagella. A total of 30 out of the 31 components described for this organelle were identified , but neither of the two strains contained a homolog of the flhE gene. Regarding the 27 components associated with type IV pilus biogenesis and regulation in Xanthomonas [32, 45, 46], fimX, pilD, pilE, pilL and pilW genes were absent in strain CITA 44, whereas strain IVIA 2626.1 sequence did not contain homologs for fimX and pilL genes.
In the genus Xanthomonas , 24 structural and regulatory components of the T3SS have been determined. They are present in the hrp gene cluster which is regulated by the master regulons HrpG and HrpX . Strain CITA 44 did not contain any of the 24 components of this gene cluster except two coding sequences which correspond to hrpG and hrpX homologs. The absence of T3SS has also been reported for another X. arboricola strain isolated from barley as well as for X. cannabis [10, 48]. The absence of the genes hrcC, hrcJ, hrcN, hrcR, hrcS, hrcT, hrcU, hrcV, hrpB1, hrpD5 and hrpF was corroborated by conventional PCR as previously described . In the case of strain IVIA 2626.1, 22 out of the 24 components, as well as homologs for the two master regulons were present, but no homologs for hpaF and hrpB5 were found. Homologs for these two genes were also absent in all the genome sequences of X. arboricola publicly available. Sixty T3Es described in genus Xanthomonas were absent in strain CITA 44 and absence of 21 of them, identified in X. arboricola pv. pruni, was corroborated by conventional PCR using specific primers . On the other hand, strain IVIA 2626.1 contained 22 T3Es, 21 of them were described previously in other X. arboricola pv. pruni strains . In addition to these effectors, a homolog of xopAQ was found. Both strains contained all 12 components associated with Agrobacterium tumefaciens [46, 49] VirB/VirD4 T4SS . Additionally, strain IVIA 2626.1 harbored a gene cluster homologous to the type four conjugation cluster (tfc). This cluster is composed by 24 genes associated with the expression of a conjugative pilus which is involved in the propagation of genomic islands . In strain IVIA 2626.1, 17 out of the 24 genes associated with the T4SS were found and, within them, tfc2, tfc4, tfc12, tfc14, tfc16, tfc22 and tfc23 were identified as the core components required for the functioning of this T4SS .
An additional feature of the X. arboricola pv. pruni sequence is the presence of the plasmid pXap41 (41,102 Kbp) . This plasmid is exclusively in X. arboricola pv. pruni strains and is associated with virulence because it contains some T3Es such as XopE3. Genome alignment of the plasmid pXap41 nucleotide sequence and the draft genome sequence for strain IVIA 2626.1 showed a region of 41.1 Kbp which was 99.90 % similar to the pXap41 plasmid of X. arboricola pv. pruni strain CFBP 5530. Conversely, no sequence region in the strain CITA 44 draft genome was similar to this plasmid. Negative results in the amplification of the genes repA1, repA2 and mobC associated with pXap41  confirmed the absence of this plasmid in strain CITA 44.
Here we report and describe the draft genome sequence for two X. arboricola strains, CITA 44 and IVIA 2626.1, isolated from Prunus in Spain and associated with bacterial spot of stone fruits and almond by PCR protocols for identification of this pathovar [51, 52]. The phenotype of these two strains varied for motility and virulence. Initial genomic analysis identified several differences associated with motility (Type IV pilus) and virulence (T3SS, T3Es and T4SS), including the presence of the putative virulence plasmid pXap41 only in X. arboricola pv. pruni IVIA 2626.1 and the absence of the T3SS, T3Es and the plasmid pXap41 in the avirulent strain CITA 44. All these features make the avirulent strain a candidate for comparative studies to elucidate the molecular processes associated with the plant host interaction and virulence for strains of X. arboricola on Prunus species. Likewise, comparative genomic studies with related strains could provide target sequences for design of molecular diagnostics for the different pathovars of X. arboricola , as well as to differentiate between virulent and avirulent strains. Further functional studies will also provide insights into the pathogenesis process for X. arboricola strains associated with bacterial spot of stone fruits and almond.
Vauterin L, Hoste B, Kersters K, Swings J. Reclassification of Xanthomonas. Int J Syst Bacteriol. 1995;45:472–89.
Bull CT, De Boer SH, Denny TP, Firrao G, Fischer-Le Saux M, Saddler GS, et al. Comprehensive list of names of plant pathogenic bacteria, 1980–2007. Eur J Plant Pathol. 2010;92:551–92.
Fischer-Le Saux M, Bonneau S, Essakhi S, Manceau C, Jacques MA. Aggressive emerging pathovars of Xanthomonas arboricola represent widespread epidemic clones that are distinct from poorly pathogenic strains, as revealed by multilocus sequence typing. Appl Environ Microbiol. 2015;81:4651–68.
Scortichini M, Rossi MP, Marchesi U. Genetic, phenotypic and pathogenic diversity of Xanthomonas arboricola pv. corylina strains question the representative nature of the type strain. Plant Pathol. 2002;51:374–81.
Lamichhane JR. Xanthomonas arboricola diseases of stone fruit, almond, and walnut trees: Progress toward understanding and management. Plant Dis. 2014;98:1600–10.
Efsa PLH. Panel (EFSA Panel on Plant Health). Scientific opinion on pest categorisation of Xanthomonas arboricola pv. pruni (Smith) Dye. EFSA. Journal. 2014;12:3857.
Palacio-Bielsa A, Cambra M, Cubero J, Garita-Cambronero J, Roselló M, López MM. La mancha bacteriana de los frutales de hueso y del almendro (Xanthomonas arboricola pv. pruni) una grave enfermedad emergente en España. Phytoma-España. 2014;259:36–42.
Garita-Cambronero J, Sena M, Sabuquillo P, Bianco MI, Ferragud E, Redondo C, et al. Early steps in the infection process in two Xanthomonas spp. models: Chemotaxis and biofilm formation. Acta Phytopathol Sin. 2013;43 Suppl 1:419.
Garita-Cambronero J, Sena-Vélez M, Palacio-Bielsa A, Cubero J. Draft genome sequence of Xanthomonas arboricola pv. pruni strain Xap33, causal agent of bacterial spot disease on almond. Genome Announc. 2014;2:e00440–14.
Ignatov AN, Kyrova EI, Vinogradova SV, Kamionskaya AM, Schaad NW, Luster DG. Draft genome sequence of Xanthomonas arboricola strain 3004, a causal agent of bacterial disease on barley. Genome Announc. 2015;3:e01572–14.
Ibarra-Caballero J, Zerillo MM, Snelling J, Boucher C, Tisserat N. Genome sequence of Xanthomonas arboricola pv. corylina, isolated from Turkish Filbert in Colorado. Genome Announc. 2013;1:e00246–13.
Pothier JF, Vorhölter FJ, Blom J, Goesmann A, Pühler A, Smits TH, et al. The ubiquitous plasmid pXap41 in the invasive phytopathogen Xanthomonas arboricola pv. pruni: Complete sequence and comparative genomic analysis. FEMS Microbiol Lett. 2011;323:52–60.
Burch AY, Shimada BK, Browne PJ, Lindow SE. Novel high-throughput detection method to assess bacterial surfactant production. Applied Environ Microbiol. 2010;76:5363–72.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis version 6.0.. Mol Biol and Evol. 2013;30:2725–9.
Trébaol G, Manceau C, Tirilly Y, Boury S. Assessment of the genetic giversity among strains of Xanthomonas cynarae by randomly amplified polymorphic DNA analysis and development of specific characterized amplified regions for the rapid tdentification of X. cynarae. Appl Environ Microbiol. 2001;67:3379–84.
Siciliano F, Torres P, Sendín L, Bermejo C, Filippone P, Vellice G, et al. Analysis of the molecular basis of Xanthomonas axonopodis pv. citri pathogenesis in Citrus limon. Electron J Biotechn. 2006;9:3.
MacWilliams, Maria P. and Liao, Min K. Luria Broth (LB) and Luria Agar (LA) Media and Their Uses Protocol. In: ASM MicrobeLibrary. American Society for Microbiology. 2006. http://www.microbelibrary.org/component/resource/laboratory-test/3031-luria-broth-lb-and-luria-agar-la-media-and-their-uses-protocol. Accessed 23 Sept 2015.
Sena-Vélez M, Redondo C, Gell I, Ferragud E, Johnson E, Graham JH, et al. Biofilm formation and motility of Xanthomonas strains with different citrus host range. Plant Pathol. 2015;64:767–75.
Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, et al. The minimum information about a genome sequence (MIGS) specification. Nat Biotech. 2008;26:541–7.
Chevreux B, Wetter T, Suhai S. Genome sequence assembly using trace signals and additional sequence information. In: Wingender E, editor. Proceedings of the German Conference on Bioinformatics: 4–6 October 1999; Hannover. Braunschweig: GBF-Braunschweig; 1999. p. 45–56.
Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: rapid annotations using subsystems technology. BMC genomics. 2008;9:1–15.
Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 1999;27:4636–41.
Lagesen K, Hallin P, Rødland EA, Stærfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35:3100–8.
Schattner P, Brooks AN, Lowe TM. The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res. 2005;33 Suppl 2:W686–89.
Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: Discriminating signal peptides from transmembrane regions. Nat Meth. 2011;8:785–6.
Krogh A, Larsson B, von Heijne G, Sonnhammer ELL. Predicting transmembrane protein topology with a hidden markov model: Application to complete genomes. J Mol Biol. 2001;305:567–80.
Tatusov RL, Galperin MY, Natale DA, Koonin EV. The COG database: A tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000;28:33–6.
Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY, Eddy SR, et al. Pfam: The protein families database. Nucleic Acids Res. 2014;42:D222–30.
Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, et al. CDD: NCBI’s conserved domain database. Nucleic Acids Res. 2014;43:D222–26.
Blocker A, Komoriya K, Aizawa SI. Type III secretion systems and bacterial flagella: Insights into their function from structural similarities. Proc Natl Acad Sci USA. 2003;100:3027–30.
Chevance FFV, Hughes KT. Coordinating assembly of a bacterial macromolecular machine. Nat Rev Micro. 2008;6:455–65.
Dunger G, Guzzo CR, Andrade MO, Jones JB, Farah CS. Xanthomonas citri subsp. citri Type IV pilus is required for twitching motility, biofilm development, and adherence. MPMI. 2014;27:1132–47.
Büttner D, Bonas U. Regulation and secretion of Xanthomonas virulence factors. FEMS Microbiol Rev. 2010;34:107–33.
Abby SS, Rocha EPC. The non-flagellar type III secretion system evolved from the bacterial flagellum and diversified into host-cell adapted systems. PLoS Genet. 2012;8:e1002983.
White FF, Potnis N, Jones JB, Koebnik R. The type III effectors of Xanthomonas. Mol Plant Pathol. 2009;10:749–66.
Hajri A, Pothier JF, Fischer-Le Saux M, Bonneau S, Poussier S, Boureau T, et al. Type three effector genes distribution and sequence analysis provides new insights into pathogenicity of plant pathogenic Xanthomonas arboricola. Appl Environ Microbiol. 2011;78:371–84.
Guglielmini J, Néron B, Abby SS, Garcillán-Barcia MP, la Cruz F, Rocha EPC. Key components of the eight classes of type IV secretion systems involved in bacterial conjugation or protein secretion. Nucleic Acids Res. 2014;42:5715–27.
Waksman G, Orlova EV. Structural organisation of the type IV secretion systems. Curr Opin Microbiol. 2014;17:24–31.
Christie PJ, Whitaker N, González-Rivera C. Mechanism and structure of the bacterial type IV secretion systems. BBA-Mol Cell Res. 1843;2014:1578–91.
Darling AE, Mau B, Perna NT. ProgressiveMauve: Multiple genome alignment with gene gain, loss and rearrangement. PLoS ONE. 2010;5:e11147.
Martínez-García PM, Ramos C, Rodríguez-Palenzuela P. T346Hunter: A novel web-based tool for the prediction of type III, type IV and type VI secretion systems in bacterial genomes. PLoS ONE. 2015;10:e0119317.
Jehl MA, Arnold R, Rattei T. Effective-a database of predicted secreted bacterial proteins. Nucleic Acids Res. 2011;39:D591–95.
Bi D, Liu L, Tai C, Deng Z, Rajakumar K, Ou HY. SecReT4: A web-based bacterial type IV secretion system resource. Nucleic Acids Res. 2013;41:D660–65.
Galperin MY, Makarova KS, Wolf YI, Koonin EV. Expanded microbial genome coverage and improved protein family annotation in the COG database. Nucleic Acids Res. 2015;43:D261–69.
Dowson WJ. On the systematic position and generic names of the gram negative bacterial plant pathogens. Zentralblatt für Bakteriologie, Parasitenkunde, Infektionskrankheiten und Hygiene Abteilung II. 1939;100:177–93.
Skerman VBD, McGowan V, Sneath PHA. Approved Lists of bacterial names. In Int J Syst Bacteriol. 1980;30:225–420.
Guo Y, Figueiredo F, Jones J, Wang N. HrpG and HrpX play global roles in coordinating different virulence traits of Xanthomonas axonopodis pv. citri. MPMI. 2011;24:649–61.
Jacobs JM, Pesce C, Lefeuvre P, Koebnik R. Comparative genomics of a cannabis pathogen reveals insight into the evolution of pathogenicity in Xanthomonas. Front Plant Sci. 2015;6:431.
Conn HJ. Validity of the Genus Alcaligenes. J Bacteriol. 1942;44:353–60.
Juhas M, Crook DW, Dimopoulou ID, Lunter G, Harding RM, Ferguson DJP, et al. Novel type IV secretion system involved in propagation of genomic islands. J Bacteriol. 2007;189:761–71.
Palacio-Bielsa A, Cubero J, Cambra MA, Collados R, Berruete IM, López MM. Development of an efficient real-time quantitative PCR protocol for detection of Xanthomonas arboricola pv. pruni in Prunus species. Appl Environ Microbiol. 2011;77:89–97.
Pagani M.C. An ABC transporter protein and molecular diagnoses of Xanthomonas arboricola pv. pruni causing bacterial spot of stone fruits. Ph.D. Thesis. North Carolina University, Raleigh, NC, USA. 2004;8-27. http://repository.lib.ncsu.edu/ir/bitstream/1840.16/4540/1/etd.pdf.
Field D, Amaral-Zettler L, Cochrane G, Cole JR, Dawyndt P, Garrity GM, et al. The Genomic Standards Consortium. PLoS Biol. 2011;9:e1001088.
Woese CR, Kandler O, Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA. 1990;87:4576–9.
Garrity GM, Bell JA, Lilburn T. Phylum XIV. Proteobacteria phyl. nov. In: Garrity GM, Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. Volume 2 (Part B). 2nd ed. New York: Springer; 2005. p. 1.
Garrity GM, Bell JA, Lilburn T. Class III. Gammaproteobacteria class. nov. In: Brenner DJ, Krieg NR, Staley JT, Garrity GM, editors. Bergey’s Manual of Systematic Bacteriology. Volume 2, part B. 2nd ed. New York: Springer; 2005. p. 1.
Validation List no. 106. Validation of publication of new names and new combinations previously effectively published outside the IJSEM. Int J Syst Evol Microbiol. 2005;55:2235–8.
Williams KP, Kelly DP. Proposal for a new class within the phylum Proteobacteria, Acidithiobacillia classis nov., with the type order Acidithiobacillales, and emended description of the class Gammaproteobacteria. Int J Syst Evol Micr. 2013;63:2901–6.
Christensen P, Cook FD. Lysobacter, a new genus of nonfruiting, gliding bacteria with a high base ratio. Int J Syst Bacteriol. 1978;28:367–93.
Naushad S, Adeolu M, Wong S, Sohail M, Schellhorn H, Gupta R. A phylogenomic and molecular marker based taxonomic framework for the order Xanthomonadales: proposal to transfer the families Algiphilaceae and Solimonadaceae to the order Nevskiales ord. nov. and to create a new family within the order Xanthomonadales, the family Rhodanobacteraceae fam. nov., containing the genus Rhodanobacter and its closest relatives. A van Leeuw J Mricrob. 2015;107:467–85.
Van den Mooter M, Swings J. Numerical analysis of 295 phenotypic features of 266 Xanthomonas strains and related strains and an improved taxonomy of the genus. Int J Syst Bacteriol. 1990;40:348–69.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene Ontology: Tool for the unification of biology. Nat Genet. 2000;25:25–9.
Kraiselburd I, Alet AI, Tondo ML, Petrocelli S, Daurelio LD, Monzón J, et al. A LOV protein modulates the physiological attributes of Xanthomonas axonopodis pv. citri relevant for host plant colonization. PLoS ONE. 2012;7:e38226.
List Editor. Validation list no. 115. List of new names and new combinations previously effectively, but no validly, published. Int J Syst Evol Microbiol. 2007;57:893–7.
Gabriel DW, Kingsley MT, Hunter JE, Gottwald T. Reinstatement of Xanthomonas citri (ex Hasse) and X. phaseoli (ex Smith) to species and reclassification of all X. campestris pv. citri strains. Int J Syst Bacteriol. 1989;39:14–22.
Darling AC, Mau B, Blattner FR, Perna NT. Mauve: Multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004;14:1394–403.
Stothard P, Wishart DS. Circular genome visualization and exploration using CGView. Bioinformatics. 2005;21:537–9.
This work was supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) grants projects RTA2011-00140-C03-02, RTA2014-00018-C02-01 and XAPDIAG EUPHRESCO II project. JGC held a PhD fellowship from the Spanish Government (Ministerio de Educación, Cultura y Deporte fellowship FPU12/01000). We like to express our gratitude to Dr. James H. Graham from the University of Florida, Citrus Research and Education Center (CREC), for the scientific and English revision of this manuscript.
The authors declare that they have no competing interests.
JGC performed the experiments, the annotation and the sequence analysis and homology searches. JCD and JGC conceived and designed the experiments. APB and MML participate in the study design, coordination and helped to draft the manuscript. JGC, APB, MML and JCD wrote this manuscript. All the authors read and approved the final manuscript.