Draft genome sequence of Xanthomonas fragariaereveals reductive evolution and distinct virulence-related gene content
© Vandroemme et al.; licensee BioMed Central Ltd. 2013
Received: 28 November 2012
Accepted: 20 November 2013
Published: 25 November 2013
Xanthomonas fragariae (Xf) is a bacterial strawberry pathogen and an A2 quarantine organism on strawberry planting stock in the EU. It is taxonomically and metabolically distinct within the genus Xanthomonas, and known for its host specificity. As part of a broader pathogenicity study, the genome of a Belgian, virulent Xf strain (LMG 25863) was assembled to draft status and examined for its pathogenicity related gene content.
The Xf draft genome (4.2 Mb) was considerably smaller than most known Xanthomonas genomes (~5 Mb). Only half of the genes coding for TonB-dependent transporters and cell-wall degrading enzymes that are typically present in other Xanthomonas genomes, were found in Xf. Other missing genes/regions with a possible impact on its plant-host interaction were: i) the three loci for xylan degradation and metabolism, ii) a locus coding for a ß-ketoadipate phenolics catabolism pathway, iii) xcs, one of two Type II Secretion System coding regions in Xanthomonas, and iv) the genes coding for the glyoxylate shunt pathway. Conversely, the Xf genome revealed a high content of externally derived DNA and several uncommon, possibly virulence-related features: a Type VI Secretion System, a second Type IV Secretion System and a distinct Type III Secretion System effector repertoire comprised of multiple rare effectors and several putative new ones.
The draft genome sequence of LMG 25863 confirms the distinct phylogenetic position of Xf within the genus Xanthomonas and reveals a patchwork of both lost and newly acquired genomic features. These features may help explain the specific, mostly endophytic association of Xf with the strawberry plant.
Xanthomonas fragariae (Xf) is a bacterial strawberry pathogen and the cause of angular leaf spot. It was first described in the United States in 1962  and has since spread globally. Under favourable conditions the pathogen may cause significant damage to both plant stock and strawberry production . Xf is a quarantine pest on planting stock within the EU , which may explain why this generally considered mild pathogen has remained at the heart of scientific and legislative debate for decades. Xf is a distinct and homogeneous species within the otherwise complex and highly dynamic genus Xanthomonas[4–7]. A certain degree of infraspecific diversity within Xf has been observed, but in general it is considered as a coherent and stable species [8–10].
Unlike its clear taxonomic position, the disease-related capabilities of Xf are still obscure. One well-established characteristic of Xf is its narrow host range: Fragaria spp. are the only natural hosts, although close relatives of Fragaria, such as Potentilla fruticosa and P. glandulosa, showed symptoms after artificial inoculation and therefore are considered potential hosts . Another, poorly characterized feature of Xf is its symptomless persistence in strawberry crops , which holds significant relevance for Xf as quarantine organism in strawberry planting stock. Molecular testing repeatedly demonstrated Xf presence in symptomless rhizomes of strawberry plants intended for planting [13, 14]. Knowledge on the in planta movement of Xf is limited:so far, only one study presented experimental evidence for the endophytic spread of Xf down from infected strawberry leaves to the rhizome and to newly emerging runners and daughter plants . Xf is a challenging organism to study because of its fastidious nature on most common growth media , and its rapidly declining viability after contact with strawberry leaf extracts . Moreover, Xf appeared insusceptible to genetic manipulation, which hampered our efforts in developing fluorescent and functional mutants (unpublished results).
A whole genome sequence of Xf can provide insight in its life style and help solve some of the technical problems it presents in the laboratory. Recent advances in sequencing technology and bioinformatics, together with emerging commercial whole genome sequencing services, have resulted in rapid and cost-effective means of generating draft genomes fit for most plant-pathology related studies . Also within Xanthomonas, multiple genome sequences are available and already provided interesting insights in the most common pathogenicity determinants of the genus [18, 19]. One of the final technical challenges associated with next-generation sequencing techniques is the presence of repetitive genomic sequences . Multiple paired-read datasets with varying insert sizes are often used to resolve assembly ambiguities associated with these repetitive sequences, or at least to bridge sequence gaps by concatenating related contigs into larger scaffolds. In addition, several software tools providing automatic scaffold gap-closure have recently been released: Gapcloser , IMAGE  and Gapfiller .
The aim of the current study was to generate a draft genome sequence of a Belgian, virulent Xf strain (LMG 25863) and to analyse its virulence-related gene content by comparison to available Xanthomonas whole-genome sequences. Two commercially obtained paired-read datasets were combined, and an automatic gap-closure algorithm was applied, to overcome encountered assembly problems related to repetitive DNA. Here, we present the resulting draft genome sequence of Xf LMG 25863 and the observed virulence-related features.
Results and discussion
Repetitive DNA content complicates genome assembly
Main characteristics of initial de novo and final draft genome assembly of X. fragariae LMG 25863
Final draft genome sequence
Contigs (> 200 bp)
Total Contig Size (bp)
N50 contig number a
N50 length (bp) b
Mapped Reads (% of total)
Reads in Aligned Pairs (% of total)
Genomes used in this study
Genome size (Mb)
X. albilineans GPE PC73
X. arboricola LMG 19145
X. arboricola LMG 19146
X. arboricola pv. pruni LMG 25862
Apricot, Plum & peach
X. axonopodis (citri) pv. citri 306
Bacterial Canker A
X. axonopodis pv. citrumelonis F1 (FL 1195)
X. axonopodis pv. punicae LMG 859
X. campestris (vasicola) pv. musacearum NCPPB 4381
X. campestris (vasicola) pv. vasculorum NCPPB 702
X. campestris pv. campestris ATCC 33913
X. campestris pv. campestris 8004
X. campestris pv. campestris B100
X. campestris pv. raphani 756C
X. campestris pv. vesicatoria (X. euvesicatoria) 85-10
Pepper & tomato
Bacterial Spot A
X. citri pv. mangiferaeindicae LMG 941
X. fragariae LMG 25863
Angular Leaf Spot
X. fuscans pv. aurantifolii ICPB 11122
Bacterial Canker B
X. fuscans pv. aurantifolii ICPB 10535
Bacterial Canker C
X. gardneri ATCC 19865
Pepper & tomato
Bacterial Spot D
X. oryzae pv. oryzae KACC 10331
X. oryzae pv. oryzae MAFF 311018
X. oryzae pv. oryzae PXO 99A
X. oryzae pv. oryzicola BLS256
X. perforans 91-118
Bacterial Spot C
X. sacchari NCPPB 4393
X. vesicatoria ATCC 35937
Pepper & tomato
Bacterial Spot B
Phylogenetic affiliation of X. fragariae to other Xanthomonasspecies
Xf reveals genome reduction similar to X. oryzae and X. albilineans
The reduced Xf genome has the major virulence-related gene regions
Major virulence related gene regions in the 5 Xanthomonas genomes compared in the EDGAR analysis
Noteworthy gene-regions missing in X. fragariae LMG 25863
Phenolics Degradation I
Phenolics Degradation II
Xylan degradation I
Xylan degradation III
Xylan degradation III
Although some absent gene-regions in Xf may have virulence-related implications, the genome reduction in Xf seems to weaken nutritional and adaptive flexibility rather than clear virulence functions. For example, the absence of all three xylan degradation loci and the ß-ketoadipate pathway may indicate that Xf is unable to respectively degrade xylan and metabolize the phenolic components of lignin, two important elements of the secondary plant cell wall . Perhaps, the opinion that primarily soil bacteria have been associated with lignin degradation , might suggest that the main role of the ß-ketoadipate pathway lays in saprophytic survival. Likewise, though the glyoxylate shunt pathway has been linked to successful symbiotic and pathogenic plant-bacterial interactions, it does so by increasing metabolic fitness through growth on C2-compounds . Potassium is another important nutritional element, crucial for cell turgor maintenance, activation of cellular enzymes and pH homeostasis. The kdp potassium transport system is widely distributed among bacteria and serves as an emergency K+-scavenging system that is only expressed and activated under extreme environmental stress . Maybe these missing functions are redundant for Xf’s existence in the strawberry leaf apoplast. Among the 26 analysed Xanthomonas genomes, the xylan degradation pathway, the sdk potassium transport system and the entire phenolics degradation pathway were uniquely missing in Xf.
The Xf genome has a reduced TonB-dependent transporter set
Xf has a reduced plant cell wall degrading enzyme set
Despite the contribution of CWDEs to virulence, the reduced set of Xf does not necessarily make it a lesser pathogen. Smaller CWDE repertoires are typically found in biotrophic pathogens, who rely on precise breaching of the host cell wall during infection instead of extensive tissue destruction observed for necrotrophic pathogens . One potential explanation for the reduced CWDE repertoire of Xf may be found in the concurrent absence of the ß-ketoadipate phenolics degradation pathway: strawberry plant tissue is rich in phenolics  and many contribute to plant defence as phytoanticipins or phytoalexins, which are often released when plant cell integrity is compromised . Therefore, one could hypothesize that one way for Xf to survive long-term residence in its potentially toxic host is to avoid extensive tissue damage.
Xf exhibits a distinct T3SE repertoire and several putatively new effectors
Known type III secretion effectors of X. fragariae LMG 25863
Best match for Xf- sequence
Worst match among other sequences
[Genbank:O1K_11182, Genbank:O1K_11197, Genbank:O1K_11202, Genbank:O1K_11207, Genbank:O1K_11212]; Contig64; Contig67
[Genbank:O1K_14315]; Contig24 (1..5097); Contig57; Contig92 (89708..96529)
Potential new type III secretion effectors found in X. fragariae LMG 25863
Most closely related knownXanthomonasT3SE
Best genbank match
Moved start-codon compared to PGAAP annotation
Best match for Xf
Worst match amongst the sequences within the effector class
Similarity with Xf-sequence
147 bp to 5' side
492 bp to 5' side
507 bp to 5' side
[Genbank:O1K_01569, Genbank: O1K_01579]a
975 bp to 5' side
399 bp to 5' side
453 bp to 5' side
633 bp to 5' side
342 bp to 5' side
891 bp to 5' side
246 bp to 5' side
Xf harbours a Type VI secretion system similar to X. oryzae
Evidence of considerable horizontal gene transfer and a CRISPR in Xf
In addition to some virulence genes that are possibly acquired by Horizontal Gene Transfer (HGT) and IS elements, other evidence of HGT-exposure could be found in the Xf genome. The IS-content appeared exceptionally abundant in Xf. While IS abundance is a common feature of Xanthomonas genomes, exceptionally high IS content was previously reported for the three Xoo strains and the Xoc strain [32, 36–38]. There, it was interpreted as a result of the consistent association of these strains with rice: the stable environment would have alleviated the selective pressure of many genes, allowing their disruption by IS. A similar process could be envisioned for Xf. Conversely, it has also been described as an important source of genome plasticity in X. oryzae and a cause of the genotypic diversity within the species [32, 38]. This seems to conflict with the restricted genotypic diversity reported for Xf. Perhaps the relatively young practice of formal plant breeding in strawberry cultivation (18th-19th century AD)  compared to the ancient domestication of rice (7000–4000 BC)  has to be considered.
Toxin-antitoxin modules in X. fragariae LMG 25863
Another HGT-related CDS in Xf with a potential virulence related function, [Genbank:O1K_06242], coded for a 2485aa putative Repeats-in-toxin (RTX) exoprotein . Its low%GC (54%) and the presence of IS elements directly up and downstream seem to indicate that it was acquired through HGT.
Finally, the Xf genome revealed a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-region comprised of 6 CRISPR associated (cas) genes of the so-called Ypest (“Yersinia pestis”) subtype ([Genbank:O1K_01919] to [Genbank:O1K_01944]), a 121 bp long AT-rich leader sequence and a CRISPR containing 36 identical repeats and 36 spacers. A second, smaller locus containing an additional 4 repeats and 3 spacers was also found, although the last repeat in this locus was degenerated. Among all other studied Xanthomonas genomes, cas-genes of the Ypest-subtype were also found in Xalb and X. campestris pv. raphani 756C (Xcr). Moreover, the associated CRISPR repeat sequence in Xf (GTTCACTGCCGCGTAGGCAGCTCAGAAA) was identical to that of Xcr, and diverged only one nucleotide from that of Xalb (GTTCACTGCCGTGTAGGCAGCTCAGAAA). CRISPR regions were recently recognized as a prokaryotic adaptive immune system against invading DNA molecules, with functional analogy to the RNA interference (RNAi) pathways in eukaryotes .
The draft genome sequence of Xf provided valuable insight in its general and more specific pathogenicity-related gene content. Although the current total contig size of 4.2 Mb is not definite, the sheer amount of missing gene homologs in Xf is sufficient evidence for a significant genome reduction. A similar “convergent genome erosion” was already reported for X. albilineans and X. oryzae pv. oryzae, and ascribed to their restricted lifestyle within the xylem vessels of their hosts . A similar genome reduction was found here in Xf and also in X. oryzae pv. oryzicola BLS256, which are two non-vascular leaf pathogens. Therefore, it may be more accurate to ascribe the convergent genome reduction of these Xanthomonas species to their endophytic lifestyle and typically to their commitment to a single host. Similar to earlier comparative genomic studies within Xanthomonas[30, 45], we were unable to find clear determinants for host or tissue specificity. Perhaps this specificity is the result of a more complex interplay between different genes or, of a subtle sequence variation within a small set of conserved genes. Alternatively, clear host or tissue determinants may still remain hidden within the substantial group of proteins for which we currently have no clear molecular function, or in uncharacterised functional RNAs.
Based on the data presented here, one could hypothesize an evolutionary process for Xf that is reminiscent of the model that was recently presented for some dangerous epidemic bacteria of humans . During an initial period of intense horizontal gene transfer experienced by the more generalist ancestor of Xf, acquirement of certain heterologous host-specificity factors would have allowed it to colonize the strawberry leaf apoplast and thus escape antagonists and environmental threats. This transition from a dynamic to a stable environment would subsequently have triggered the observed genome reduction: useless or redundant features, especially metabolic, perceptive and regulatory functions, were allowed to degrade and eventually were lost. At some point, the progressing genome erosion resulted in the effective metabolic “entrapment” of Xf within the strawberry plant, excluding it from other hosts or more general epiphytic or saprophytic lifestyles. Because of Xf’s increasing spatial and phylogenetic isolation, the initially intense horizontal gene transfer would have abated, a process that was perhaps hastened by the acquirement of the CRISPR region. Meanwhile, mobile genetic elements which conferred a selective advantage to Xf would have become permanently incorporated in the genome. This evolutionary process would finally have resulted in the genotypical and phenotypical distinct, mainly endophytic, biotrophic and strawberry-specific Xf known today. The eventual necrosis of the typical water-soaked angular leaf spots associated with Xf would not be in conflict with this hypothesis: it could be the manifestation of an eventual breakthrough of the plant defence, or merely the collapse of an overburdened plant cellular system.
Of course, many of the in-silico based hypotheses presented here should be tested and confirmed by further experimental data. In our opinion, the most appealing matters for future research are i) the molecular and functional characterization of the putatively new PsvA-like T3SEs, ii) the exact function of the T6SS in Xf and other Xanthomonas, and iii) the delicate endophytic existence of Xf, sensitive to toxic compounds inside the strawberry plant cells.
Strain selection, culture conditions and DNA preparation
The selected Xf strain, LMG 25863, was isolated as GBBC-Xf 920 in 2002 from clear angular leaf spots on strawberry leaves at the Institute for Agricultural and Fisheries Research (ILVO) in Belgium. Since then, it has been applied in the development of a real-time PCR detection method for Xf , served as Xf reference in a study of X. arboricola pv. fragariae and as parental strain in the development of a green fluorescent Xf mutant (unpublished). It was deposited at the BCCM-LMG culture collection at the time of whole genome sequencing. For genomic DNA preparation, the cryogenically stored Xf strain was resuscitated by incubation on Wilbrinck-N agar medium  at 28°C for 96 h. A single colony of this culture was then transferred to fresh Wilbrink-N agar plates and again incubated at 28°C for 48 h. From these cultures, a total of 30 μg DNA with a concentration of at least 100 ng/μl and an OD260:280 rating between 1.8 and 2.0 was prepared using the Gentra Puregene Cell Kit (QIAGEN Benelux B.V., Venlo, The Netherlands), according to the manufacturer’s instructions. Quantity and quality of the extracted DNA was checked using the NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).
Sequencing, draft genome assembly and annotation
Custom DNA library preparation and Illumina-sequencing was performed by Baseclear N.V., Leiden, The Netherlands. A first Paired-End (PE) DNA library with an a mean insert size of 375 bp was sequenced with 50 bp reads on an Illumina Genome Analyzer IIx (Illumina Inc., San-Diego, USA). A second, Mate-Paired (MP) DNA library with a mean insert size of 5100 bp was sequenced with 75 bp reads on an Illumina Hiseq2000 (Illumina Inc.), but only the first 50 bp were used to avoid chimeric reads.
The received raw PE and MP read sets were quality trimmed in CLC Bio v4.0 (CLC bio, Aarhus, Denmark) using a Phred quality cut-off score of 20. An initial de novo assembly was performed in CLC Bio v4.0 using only the PE reads, and all contigs shorter than 200 bp were discarded. This assembly was scaffolded in SSPACE Premium v2.0  using MP reads and processed with the Gapcloser v1.12 tool of the SOAP genome assembly software . Gapfiller and IMAGE were not used because the former was not yet freely available and the latter could not be operated as intended. Because Gapcloser did not recognize the Hiseq2000 file parsing of the MP dataset, only the PE data was used. Finally, the draft genome sequence was manually edited with the Editseq tool of DNAStar Lasergene core suite v10.0.1 (DNASTAR Inc., Madison, WI, USA). Remaining N-nucleotides in the scaffolds, introduced during scaffolding and not replaced by gapcloser, were removed from the final sequence by breaking up the scaffolds back into contigs where they were encountered. The quality of the final draft genome sequence was compared to the initial PE-based de novo assembly through comparative read-mapping in CLC Bio v4.0 using both the trimmed read sets. The final draft genome sequence of Xf was putatively annotated with the RAST v4.0 online annotation pipeline  and NCBI’s Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP; http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html).
Comparative genome analysis
The presence of possible pathogenicity-related genes in the Xf draft genome was analysed by comparison with 25 other Xanthomonas genomes (Table 2). In a first explorative screening, the gene content of the Xf genome was compared with the chromosomes X. campestris pv. campestris (Xcc) ATCC 33913, X. euvesicatoria (Xcv) 85–10, X. oryzae pv. oryzae (Xoo) KACC 10331 and X. albilineans (Xalb) GPE PC73 in EDGAR . Absence or presence of coding sequences in each genome, as reported by EDGAR, were independently confirmed by performing protein and nucleotide blast queries (as described below) in the target genomes before inclusion in this manuscript.
In a second phase, gene families of interest were examined in all 26 genomes. All genomes except three were screened using the protein and nucleotide blast tools of Genbank. Four genomes not present in Genbank were screened with the blast tool of the SEED Viewer v2.0 web-interface : draft genomes of the present Xf strain LMG 25863, two X. arboricola strains LMG 19145 and LMG 19146 , and the X. arboricola pv. pruni strain LMG 25864 (M Maes, unpublished). Based on the EDGAR results, three protein families were studied in greater detail: the TonB-dependent transporters (TBDT), the cell-wall degrading enzymes (CWDE) and the Type III secretion system effectors (T3SE).
To determine the phylogenetic position of Xf within the Xanthomonas genus, we performed in silico multi-locus sequence analysis using partial sequences of the genes gyrB, rpoD, atpD, dnaK and fyuA, according to Parkinson et al. (2009) , Young et al. (2008) , and Ngoc et al. (2010) . Sequences were retrieved from the Xf and the 25 other Xanthomonas genomes available in GenBank (Table 2). The sizes of the five partial sequences are 530 bp (gyrB), 747 bp (atpD), 940 bp (dnaK), 873 bp (rpoD) and 698 bp (fyuA), giving a total of 3788 bp for the concatenated dataset. Sequences were concatenated and aligned using the CLUSTALW algorithm . Sequence alignment, trimming, and phylogenetic analysis were performed in Mega5 . The phylogenetic tree was generated using the Maximum Composite Likelihood method for calculating distances and the Neighbor Joining algorithm for clustering , bootstrap analysis was performed using 1000 bootstrap replicates.
The TBDT repertoire of Xf was compared to that of the other 25 Xanthomonas genomes. The 72 TBDTs identified in Xcc ATCC 33913  were used as primary references. Initially, the 26 genomes were screened for homologs of these primary references using protein blast queries. In case of a negative blast result, an additional nucleotide blast with the reference coding sequence was performed to exclude false negative results. TBDTs with low protein sequence similarity (<70%) with the primary references were considered as new types and were added to the list of references. TBDTs in the more distantly related X. albilineans GPE PC73 and X. sacchari NCPPB 4393 occasionally showed ambiguous homology with more than one reference. This was resolved by reciprocal blast, after which the TBDT in question was assigned to the reference with the best blast hit score.
Cell-wall degrading enzymes
Reference sequences of different CWDE-families were retrieved from . All 26 genomes were searched for homologs of these references in a similar fashion as applied for the TBDTs.
Type III secretion effectors
References of the presently known T3SEs in Xanthomonas were retrieved from the Xanthomonas Resource website  and all 26 genomes were screened for homologs. Because of their sometimes large diversity, the protein sequences of all encountered (putative) effectors were collected and compared in Bionumerics v6.6 (Applied Maths, Sint-Martens-Latem, Belgium). For each effector class, a protein-sequence based pairwise alignment similarity matrix was calculated using the standard Bionumerics algorithm in its default settings. Candidate effectors of Xf were only considered as part of an effector class when it exhibited at least 60% pairwise similarity with at least one other entry in the matrix. Next, a binary table listing the presence or absence of at least one fully coded homolog of every known T3SE class in each of the 26 genomes was created. Truncated, frame-shifted or otherwise suspected incomplete or inactive coding sequences were interpreted as absent. When the full coding sequence could not be retrieved because of incomplete assembly, the effector was counted as present. Multiple homologs of an effector in a single genome were counted only once. This binary dataset was imported and analyzed in Bionumerics. A binary-data based distance matrix was calculated using the Hamming Distance parameter and a distance-based consensus network was calculated using the Neighbor Joining tree method in its default settings and bootstrap resampling with 1000 replications .
The raw sequence data received from Baseclear N.V. (Leiden, The Netherlands) was deposited at the Short Read Archive (SRA) of Genbank under accession numbers SRR514114 (PE dataset) and SRR514113 (MP dataset). The current draft genome sequence was deposited at Genbank under accession number AJRZ00000000 after automatic annotation by the PGAAP online annotation pipeline.
The authors would like to acknowledge Miss. Cinzia Van Malderghem at ILVO for her technical assistance during the genomic DNA preparation, and Prof. Dr. Tom Coenye of the Laboratory of Pharmaceutical Microbiology at Ghent University for his critical reading of the draft manuscript. This work was made possible through the financial support of the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT) under project nr. 070597.
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