Genomic Survey of Pathogenicity Determinants and VNTR Markers in the Cassava Bacterial Pathogen Xanthomonas axonopodis pv. Manihotis Strain CIO151

Abstract

Xanthomonas axonopodis pv. manihotis (Xam) is the causal agent of bacterial blight of cassava, which is among the main components of human diet in Africa and South America. Current information about the molecular pathogenicity factors involved in the infection process of this organism is limited. Previous studies in other bacteria in this genus suggest that advanced draft genome sequences are valuable resources for molecular studies on their interaction with plants and could provide valuable tools for diagnostics and detection. Here we have generated the first manually annotated high-quality draft genome sequence of Xam strain CIO151. Its genomic structure is similar to that of other xanthomonads, especially Xanthomonas euvesicatoria and Xanthomonas citri pv. citri species. Several putative pathogenicity factors were identified, including type III effectors, cell wall-degrading enzymes and clusters encoding protein secretion systems. Specific characteristics in this genome include changes in the xanthomonadin cluster that could explain the lack of typical yellow color in all strains of this pathovar and the presence of 50 regions in the genome with atypical nucleotide composition. The genome sequence was used to predict and evaluate 22 variable number of tandem repeat (VNTR) loci that were subsequently demonstrated as polymorphic in representative Xam strains. Our results demonstrate that Xanthomonas axonopodis pv. manihotis strain CIO151 possesses ten clusters of pathogenicity factors conserved within the genus Xanthomonas. We report 126 genes that are potentially unique to Xam, as well as potential horizontal transfer events in the history of the genome. The relation of these regions with virulence and pathogenicity could explain several aspects of the biology of this pathogen, including its ability to colonize both vascular and non-vascular tissues of cassava plants. A set of 16 robust, polymorphic VNTR loci will be useful to develop a multi-locus VNTR analysis scheme for epidemiological surveillance of this disease.

Introduction

The genus Xanthomonas comprises plant pathogens that infect a wide range of plants, including citrus, pepper, tomato, rice and others [1], [2]. In particular, Xanthomonas axonopodis pv. manihotis (Xam) is the causative agent of cassava bacterial blight (CBB; [3]), the main bacterial disease affecting cassava plants (Manihot esculenta). This disease is widespread in all places where cassava is grown, including Africa, Asia and South America [4], [5], where crop losses due to CBB have been reported to be between 12 and 100% [3]. Accordingly, the journal Molecular Plant Pathology recently listed Xam among the top 10 plant pathogenic bacteria based on its scientific and economic importance [6].

Xam is mainly a vascular pathogen and survives epiphytically until favorable conditions for infection are reached. Open wounds and stomata are major routes of pathogen infection in otherwise healthy plants [4], and the disease is transmitted between crop cycles through the use of infected cuttings [7]. Once inside the plant, bacteria colonize the mesophyll, generating angular leaf spots as one of the early symptoms. In subsequent stages in susceptible plants, pathogen population increases and reaches vascular tissues, blocking the flow of nutrients and generating a wilting process that, in severe cases, ends with the death of the plant [3]. Alternatively, when infection starts by the use of infected propagative material, it spreads immediately in the vascular tissues, leading to a rapid wilt of the plant (reviewed by [7]). Control strategies to prevent CBB spread include the use of resistant cassava varieties and pathogen-free plant cuttings [3]. Nonetheless, the molecular basis of resistance is not completely understood and it is permanently challenged by the diversity of Xam strains [8], [9]. Also, knowledge on the early determinants of disease development is limited. A better understanding of the pathogenicity mechanisms of Xam at the molecular level is urgently needed to efficiently control this disease.

Among the most important pathogenicity factors are the diverse protein secretion systems and their substrates [10]. Of special interest are type III-secreted effector proteins (T3E), which play an important role in the plant-pathogen interaction and in shaping the host range [11], [12], [13]. Moreover, conserved T3E in Xanthomonas have been proposed as an ancestral characteristic for pathogenicity and virulence inside the genus [14]. About twenty potential T3Es per genome have been identified in different Xanthomonas, with more than sixty different potential T3E found among all the bacteria of this genus [13]. More recently, 22 effector gene families were reported to be present in several genomic sequences of Xam [15]. Despite this wealth of information, in Xam, only one T3E, belonging to the TAL effector family has been reported as a virulence factor [16]. Several other elements such as exopolysaccharides (EPS) and cell wall degrading enzymes (CWDE), with attributed pathogenicity and virulence roles in other xanthomonads, might be important for Xam [17], [18]. In fact, for this particular pathogen, the synthesis of EPS has been reported as a virulence factor [19]. However, in order to comprehensively characterise the pathogenicity repertoire of plant pathogens such as Xam it is necessary to move beyond single gene approaches and to apply genomics tools. Further, genomics approaches may reveal origins of pathogenicity and virulence factors and thus contribute to our understanding of how microbial pathogenesis evolves.

Virulence factors are microbial adaptations and can arise from de novo mutations or through gene flow among populations or species. In recent years, the importance of horizontal gene transfer (HGT) events that lead to the acquisition of foreign DNA sequences has been well documented in bacteria [20], [21], [22]. Efforts to define the impact of these events in the genomic structure and variations between closely related species have been made [23]. For example, a foreign origin of the type III secretion system (T3SS) has been proposed in the genus Xanthomonas [24], and contribution of HGT to the genome composition has been measured in X. citri pv. citri str. 306 (Xac) (syn. X. citri subsp. citri) and X. campestris pv. campestris str. ATCC 33913 (XccATCC) species [23], [25]. However, the determination of HGT events in Xam, as well as their contribution to pathogenicity is still missing.

Efficient control of CBB will also critically depend on a profound knowledge of the population structure of Xam in different regions of the world. Traditionally, bacterial isolates have been typed by various fingerprinting techniques [26], [27], [28], [29], [30], [31]. Since then, multiple loci variable number of tandem repeat (VNTR) analysis (MLVA) has become increasingly popular for molecular typing of bacteria [32], [33]. MLVA is a method for molecular typing of bacterial strains that explores the natural variation in the number of tandemly repeated DNA sequences. MLVA has several advantages over other bacterial genotyping methods, such as ease of performance and portability, high reproducibility and discriminatory power, rapidity and low costs [34]. Powerful MLVA schemes are available for most important bacterial pathogens infecting humans, including Bacillus anthracis, Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa and Yersinia pestis [32], [35]. With the advent of genomics, development of powerful MLVA schemes became a straightforward procedure [34]. Consequently, the first VNTR study of a bacterial plant pathogen, Xylella fastidiosa, was published in 2001 [36], and VNTR schemes are now available for Candidatus Liberibacter asiaticus, Pseudomonas syringae and several pathovars of Xanthomonas [37], [38], [39], [40], [41].

Here, we report the first manually annotated high-quality draft genome sequence of Xam strain CIO151, obtained by 454 sequencing technology, and analysis of the impact of presumed HGT events on the gene content of Xam. We defined a set of potential pathogenicity determinants through a comparative genomic approach and report sets of genes encoding for T3E proteins, cell wall-degrading enzymes, secretion systems, among others; thus obtaining a deeper insight into the gene repertoire of Xam CIO151. This strain was selected for sequencing for several reasons: (i) QTL mapping of resistance markers effective against CIO151 has been carried out in a cassava mapping population [42]. (ii) Aggressiveness of this strain against several cassava cultivars has been measured [9]. (iii) Effects of CIO151 inoculation on gene expression of cassava have been characterized [43], [44]. This sequence provides an efficient approach to understand the pathogenicity of this organism and to develop future studies oriented at increasing the knowledge on the biology of Xam and the cassava-Xam interaction. Finally, we took advantage of this new genomic resource to develop an inexpensive and user-friendly molecular typing tool based on VNTR loci.

Results

General features

The draft genome sequence of Xanthomonas axonopodis pv. manihotis (Xam) strain CIO151 was produced with 454 sequencing technology. This strain had recently been sequenced with Illumina technology [15] for the identification of type III effectors and the determination of phylogenetic relationships of Xam strains around the world. Though adequate for these aims, the previously reported draft genome assembly was not sufficient for a comprehensive genome mining and comparative genomics. A total of 305,637 clipped base-called reads with a median read length of 359 bases and coverage of 21.9× were obtained. The genome sequence is composed of 36 scaffolds with a total length of 5.15 Mb, a gene coding capacity of 82.8% and a N50 scaffold size of 429.5 kb. The sequence has a high G+C content (65.1%), as commonly reported for the genus Xanthomonas [45]. A total of 4340 putative coding sequences (CDS), two rRNA operons, and 55 tRNA genes for all amino acids were identified ( Table 1 ). Upon automatic annotation, an international consortium of scientists with expertise on different aspects of the biology of Xanthomonas manually annotated all predicted genes. This manual annotation led to modifications of structural annotation (860 CDS) and functional annotation (3818 CDS). In addition, 369 predicted CDS were removed.

Table 1. General features of the genome of Xam CIO151.

Features of the genome assembly of Xam CIO151
Size (bp)	5,150,225
Number of scaffolds	36
G+C (%)	65.1
Insertion sequences and transposons	250
Regions with atypical composition	62
Predicted CDS	4340

The gaps on the genome sequence did not preclude the identification and analysis of conserved gene clusters and potential pathogenicity factors. Xam CIO151 shares a number of genomic features with other members of the genus. Yet, we could detect differences that could be relevant for its biology, including variations in some T3E, the xanthomonadin cluster and regions putatively associated with HGT events.

Fifteen out of the 36 assembled scaffolds, accounting for 4.82 Mb, could be mapped to the chromosome of Xanthomonas euvesicatoria and were classified as part of the chromosome (see methods, Fig. 1 ). As usual, the dnaA gene was placed at the beginning of the genome sequence and corresponds to the start of the first scaffold. Scaffold mapping resulted in an asymmetric GC skew pattern which is typical for bacterial genomes [46], thus supporting the proposed order of scaffolds. Scaffolds that could not be mapped were classified in two categories: (i) candidate plasmid sequences of small length, which showed similarity to plasmids of other xanthomonads, and (ii) sequences of unknown origin, which did not show high similarity to chromosomal or plasmid sequences of other xanthomonads. In total, 93.64% of the genome sequence was classified as chromosomal, 1.72% was classified as candidate plasmid sequences and 4.64% was classified as unknown. The proportion of candidate plasmid and chromosomal sequences is in agreement with values reported for other Xanthomonas strains, such as Xac and Xeu [25], [47].

Figure 1. Comparison of the genomic structure of Xam CIO151 with that of closely related members from the genus Xanthomonas.

Scaffolds of Xam CIO151 were ordered based on the alignment with the complete genome sequence of X. euvesicatoria, Xeu, and then genome comparisons were performed using MUMmer (A). Alignment of ordered scaffolds of Xam CIO151 with the complete genome sequences of X. axonopodis pv. citri str. 306, Xac (B); X. campestris pv. campestris str. 8004, Xcc (C); X. albilineans, Xal (D); and Xanthomonas oryzae pv. oryzae PXO99^A, Xoo (E) chromosomes. Scaffolds classified as parts of the chromosome of Xam CIO151 are shown in the y-axis. Red dots represent conserved segments while blue dots represent inverted regions.

Xam CIO151 shares a similar genome structure with Xac and Xeu

In order to evaluate the genome structure of Xam CIO151 and to compare it with the structure of other xanthomonads, global genomic alignments ( Fig. 1 ) and an analysis of collinear blocks at the intrascaffold level were performed using MAUVE software [48] (Fig. S1). Xam CIO151 structure is most similar to that of Xac and Xeu, with three small inversions ( Fig. 1A–B ). A large rearrangement was observed in the comparison between Xam CIO151 and Xcc8004 ( Fig. 1C ). Numerous rearrangements are evident between Xam CIO151 and X. oryzae pv. oryzae PXO99^A (XooPXO99A) [49] and between Xam CIO151 and X. albilineans (Xal) [50] ( Fig. 1D–E ). MAUVE alignments showed an increase in rearrangements of collinear regions in scaffolds that mapped close to the replication terminus, which supports our genome assembly. Only two scaffolds showed rearrangements in consecutive collinear regions in alignments of Xam CIO151 with Xeu and Xac, while more evident rearrangements were observed in the alignments with Xcc and Xoo (Fig. S1). Large duplications were not detected in the set of scaffolds classified as chromosomal regions. In addition, several genomic islands, not shared by both species under comparison (white regions), were also identified (Fig. S1).

Unique proteins of Xam CIO151

Differences in host range among taxa in this genus might be associated with the presence or absence of specific pathogenicity determinants in each taxon. Based on this hypothesis, we compared the predicted proteome of Xam CIO151 with those of other xanthomonads. In total, 126 proteins were identified as unique to Xam (excluding proteins encoded by incompletely sequenced genes) (Table S1). Among these 126 proteins, the most frequent matches in BLAST searches against Genbank corresponded to translated sequences from members of the Burkholderia genus. In general, a high percentage of the Xam CIO151-specific proteins (81%) were annotated as hypothetical or conserved hypothetical proteins and only seven potentially secreted proteins and four putative membrane proteins were identified (Table S1). We also evaluated the potential presence of the 126 proteins in the genome sequence of the 65 Xam strains reported by Bart and collaborators [15] using a tblastn [51]. There were 58 proteins with hits in all 65 Xam strains (covering at least 80% of the sequence and with a similarity of at least 30%). This set of unique proteins is an important starting point in the identification of elements potentially involved in the specific cassava-Xam interaction.

Horizontal gene transfer and pathogenicity islands

Identifying genomic regions potentially acquired by horizontal gene transfer (HGT) events is a key step in understanding the features that are unique to the members within a species, as well as those which contribute to divergence in a given taxon [52], [53]. Using the automatic annotation produced by iANT (integrated ANnotation Tool) [54], a set of 250 putative insertion sequences and transposable (IS/Tnp) elements (including full and fragmented sequences) were identified in all scaffolds.

Sixty-two candidate regions with atypical nucleotide composition and potentially related to HGT events were identified in the genome sequence of Xam using AlienHunter ( Table 1 ; Fig. 2 ; Table S2). The total length of predicted regions was 640 kb (including regions in putative non-chromosomal scaffolds), which corresponds to 12.4% of the total sequence of Xam CIO151. The average length of the predicted regions is 10.3 kb and twenty two regions are larger or equal to 10 kb.

Figure 2. Circular representation of the genome sequence of Xam CIO151.

From outside to inside: first circle in blue indicates CDS predicted in the positive strands for the scaffolds classified as probable chromosomal regions. Second circle in red indicates the CDS predicted in the negative strand. Red spots in the black third circle indicate the region identified with atypical nucleotide composition. The fourth circle indicates the deviation pattern from the average G+C content. Inner circle shows GC skew values, positive values are shown in purple and negative values are shown in orange. Numbers correspond to scaffold IDs.

Genomic islands resulting from HGT events generally share some characteristics, such as the unusual G+C content, the vicinity of tRNA genes and/or mobile genetic elements, among others [55]. Indeed, 33 out of the 62 regions with atypical nucleotide composition have elements similar to reported mobile sequences (all 33 regions with plasmid sequence and seven with additional prophage derived sequences); 19 are located close to predicted tRNA genes and ten chromosomal elements have both. Additionally, twelve regions cover 50.4% of scaffolds that were not identified as part of the chromosome of Xam CIO151. This finding supports the potential alien origin of those scaffolds.

Pathogenicity islands (PAIs) constitute a special class of HGT-acquired regions. In addition to the previously described characteristics of genomic islands, PAIs also contain genes associated with virulence [55]. In order to identify potential PAIs present in the genome of Xam CIO151, we scrutinized the genomic islands for genes consistent with functions in virulence.

Four potential PAIs were identified (Table S3). The first two contain genes that have been suggested in other xanthomonads to constitute putative PAIs, including a type IV secretion system and genes associated with type IV pili (pilY, pilX, pilW, pilV and fimT) [23], [47]. The third candidate PAI encodes a XopC2 effector and two fragmented versions of the XopP effector. The fourth candidate PAI contains two probable pseudogenes (due to early stop codons) similar to the fhaB adhesin family.

Mutations in the xanthomonadin cluster could explain the white phenotype of Xam

Xanthomonadins are yellow pigments, which are diagnostic for xanthomonads (Greek xanthós = yellow) [56]. A role for xanthomonadins in epiphytic survival and protection against photobiological damage has been proposed for Xcc strain B24 [57]. However, some xanthomonads lack this pigment, including Xam, X. citri pv. mangiferaeindicae and some strains of Xcc [58]. In order to detect changes that could potentially explain the white phenotype of all Xam strains, the xanthomonadin cluster (also known as pig cluster) was analyzed ( Fig. 3A ). This cluster consists of seven transcriptional units in Xcc [58] and encodes fourteen open reading frames (ORFs) in Xoo strains [59]. Four of them are required for xanthomonadin biosynthesis, including two ORFs, XanB1 (xanmn_chr15_0086) and XanB2 (xanmn_chr15_0087), located in the pigB region [57]. Based on high sequence similarity with homologs in other xanthomonads, most genes seem to be functional in the genome of Xam CIO151. Interestingly however, gene xanmn_chr15_0082, which encodes an acyl carrier protein dehydratase, appears to be non-functional in Xam, due to a frameshift at position 110 which results in a 50 amino acid protein instead of the 95 residues reported for the Xeu predicted protein ( Fig. 3A ). This gene has been reported as an important element for xanthomonadin synthesis in Xoo strains [59] and a predicted loss of function in Xam could cause the white phenotype of this bacterium. This hypothesis is supported by an analysis of 65 draft genome sequences of Xam [15], which shows that the early stop mutation is conserved among strains in the pathovar.

Figure 3. Organization of pathogenicity-related gene clusters in the Xam CIO151 genome.

Open arrows with labels indicate genes with assigned functions, black arrows indicate genes with early stop codons, open arrows without labels indicate conserved hypothetical proteins and grey arrows indicate non-conserved hypothetical proteins. Graphs above clusters show the G+C content and deviations from the average value. A. Xanthomonadin gene cluster; * indicates genes related to pigB genomic region and ** indicates genes reported as important for cluster functionality. Abbreviations used are: H = halogenase (xanmn_chr15_0075), BP = xanthomonadin biosynthesis protein (xanmn_chr15_0079), E = xanthomonadin exporter (xanmn_chr15_0373), PSP = putative secreted protein (xanmn_chr15_0080), BACPD = xanthomonadin biosynthesis acyl carrier protein dehydratase (xanmn_chr15_0082), BA = putative xanthomonadin biosynthesis acyltransferase (xanmn_chr15_0081 and xanmn_chr15_0083), BMP = putative xanthomonadin biosynthesis membrane protein (xanmn_chr15_0084), ACP = acyl carrier protein (xanmn_chr15_0085), XanB1 = putative reductase/halogenase (xanmn_chr15_0086), XanB2 = putative pteridine-dependent deoxygenase like protein (xanmn_chr15_0087), AMP-l = AMP-ligase (xanmn_chr15_0088), DP = dipeptidyl peptidase (xanmn_chr15_0090). B. Cluster implicated in xanthan production (gum). C. Regulation of pathogenicity factors (rpf) cluster. D. Type III secretion system (T3SS) cluster.

Several secretion systems, pathogenesis-associated clusters and elements involved in cell-to-cell signaling are conserved in Xam CIO151

Cell-to-cell signaling is a key bacterial mechanism for sensing population density levels through diffusible signal molecules [60]. The genome of Xam CIO151 carries the rpf (for regulation of pathogenicity factors) gene cluster that is found in all xanthomonads and encodes components governing the synthesis and perception of the diffusible signal factor DSF [61], [62]. This cluster is composed of rpfABCDEFGH genes ( Fig. 3C ; Table S4). The Rpf/DSF system regulates the synthesis of virulence factors and biofilm formation and is required for the full virulence of Xcc, Xac, Xoc, and Xoo on their hosts [63], [64], [65], [66], [67]. RpfF is responsible for the synthesis of DSF, whereas RpfC and RpfG comprise a two-component system implicated in DSF perception and signal transduction [61], [68], [69]. RpfC is a complex sensor kinase, while RpfG is a response regulator with a HD-GYP domain that acts in degradation of the second messenger cyclic di-GMP [70]. In addition to genes encoding these products, Xam CIO151 encodes for rpfH, a membrane protein related to the sensory input domain RpfC with unknown function. RpfH is present in Xeu and Xcc but absent in Xac and Xoo.

Bacteria use several secretion systems to secrete a diversity of proteins. Xam CIO151 possesses all the protein secretion systems that have been reported so far for other Gram-negative bacteria. The corresponding gene clusters show a conserved synteny to those of Xac and Xeu. The type II secretion system (T2SS) is important for the secretion of cell wall-degrading enzymes in different plant-pathogens [71], [72], [73]. Two copies of this cluster were detected in the genome of Xam CIO151. The xps cluster (xpsEFGHIJKLMND genes) was detected in a genomic region of 10.4 kb ( Table 2 & Table S5). The xcs cluster, encoding a second T2SS (xcsCDEFGHIJKLMN genes) was identified in another genomic region of 10.8 kb ( Table 2 & Table S5). This second T2SS is present in other xanthomonads of the X. axonopodis and X. campestris species [74].

Table 2. Putative pathogenicity elements identified in the genome of Xam CIO151.

Functional category	Number of related genes
Type I protein secretion system	5
Type II protein secretion system
xps cluster	11
xcs cluster	12
Type III protein secretion system (hrp cluster)	28
Type III effector proteins	28
Type IV protein secretion system	22
Type VI protein secretion system	15
Type IV pili	24
Flagellum1	32
Regulation of pathogenicity factors (rpf cluster)	8
Xanthomonadin	18
Xanthan (gum cluster)	16
Cell wall-degrading enzymes	30
Polyketide synthase (PKS)1	26
Siderophore biosynthesis1	3
Toxins1	6
Chemotaxis1	47

¹Number indicates CDS identified by key word searches in iANT.

The T3SS encoded by the hrp gene cluster is involved in the secretion and translocation of effector proteins and is a key pathogenicity factor in most xanthomonads [75]. In Xam CIO151, it is composed of twenty-eight genes, including the hpaF effector gene and a putative xopF pseudogene, in a genomic region of 27.3 kb ( Fig. 3D ; Table S6). Its G+C content is 62.8%, a value slightly lower than the average for this genome and comparable to that of other xanthomonads [74]. Xam CIO151 shares all conserved genes of the hrp cluster and is predicted to possess two non-conserved hypothetical proteins in this cluster. One of them is located between the hpaB and xopF genes, and the other one is located between hpa3 and hrpF. Whether or not these genes play a role in pathogenicity awaits experimental confirmation.

The T4SS has been reported to mediate translocation of DNA and effector proteins in bacterial interactions with other bacteria and with their eukaryotic hosts [76], [77]. Two different clusters varying in their gene organization were identified in the sequence of Xam CIO151 and they were classified according to Moreira and collaborators [78]. The first one belongs to class I (T4SS[I]). It consists of eleven genes in a genomic region of 12.5 kb and is highly similar to the T4SS from Legionella pneumophila [79]. This is reminiscent of the type IV secretion system identified in the plasmid pXCV183 of Xeu [47]. Nonetheless, the cluster present in Xam differs from that in L. pneumophila by the apparent absence of a virB7 homolog and the presence of an additional incomplete sequence similar to virB4. Interestingly, this cluster is located on a probably non-chromosomal 35-kb scaffold with atypical nucleotide composition. It has some mobile elements and is partially similar to plasmid sequences of other xanthomonads and a plasmid from Methylobacterium radiotolerans, suggesting that this cluster is located on a plasmid. The second one is composed of eleven genes in a genomic region of 12.8 kb and was classified as belonging to class IV ( Table 2 ), just as the chromosomal T4SS of Xac and XccATCC [78]. These findings are not unique to Xam CIO151, as one chromosomal and one plasmid copy of T4SS have been reported in Xac and Xeu as well [25], [47]. The difference in their organizations and the potential plasmidic origin of one of the two T4SS suggest that both secretion systems serve in distinct functions in Xam CIO151.

The versatile bacterial type VI secretion system (T6SS) is involved in interbacterial interactions, as well as symbiotic and pathogenic interactions with eukaryotic cells [80], [81] One copy of the T6SS with fifteen genes was detected in Xam CIO151, while two T6SS have been reported in Xeu and X. oryzae species [14].

The widely conserved gum cluster of xanthomonads [74], which is involved in xanthan exopolysaccharide (EPS) production [82], has been previously related to pathogenicity in Xam [19]. In Xam CIO151, the gum cluster is composed of the gumABCDEFGHIJKLMNOP genes ( Fig. 3B ; Table S7). It shows an organization comparable to that of other xanthomonads, including a tRNA gene adjacent to the cluster [74]. The G+C content of this region is 62.8%, two percentage points below the average value for the genome.

Lipopolysaccharides (LPS) are a characteristic element of bacterial outer membranes [83]. Interestingly, LPS molecules and components thereof have been classified as PAMPs (Pathogen Associated Molecular Patterns) in plant-pathogenic bacteria [84], [85]. Despite the fact that it is a conserved element in animal and plant pathogens, variations in the LPS cluster have been associated with virulence and host range [86]. The LPS gene cluster in Xam CIO151 consists of fifteen genes located in two consecutive scaffolds, with a total length of 17.2 kb. Ten of these genes are located in a region with atypical nucleotide composition (Fig. S2). The organization of the Xam CIO151 LPS locus is similar to that of Xoo strain BX08 and Xac strain 306 [87]. However, the LPS cluster of Xam CIO151 is distinct since it shares a fragment of an IS1404 element with Xoo but is otherwise more similar in the order and number of genes with Xac (Fig. S2). Recently, very simlar LPS gene clusters have also been found in the banana pathogen X. campestris pv. musacearum strain NCPPB 4381 and in the pomegranate pathogen X. axonopodis pv. punicae strain LMG 859 [88], [89].

The Xa21 gene in rice recognizes an avirulence determinant present in Xoo, resulting in the elicitation of a strong defense response and suppression of pathogen growth. The presence and potential functionality of the genes required for avrxa21 activity (rax) was assessed in the genome of Xam CIO151. RaxH and RaxR, the two component regulatory system important for this activity, seems to be functional in Xam CIO151 (xanmn_chr10_0493 and xanmn_chr10_0494, respectively). RaxA and RaxC, a part of the Type I secretion system required for this activity also appear to be functional in Xam CIO151 (xanmn_chr10_0509 and xanmn_chr11_0184, respectively). However, the raxB gene (xanmn_chr10_0508), encoding the ATP-binding cassette transporter of the T1SS has a frameshift, resulting in a fragment encoding only 40% of the predicted functional protein, and suggesting a loss of function of this gene. This observation was confirmed in the 65 available genomes of Xam, demonstrating that it is a characteristic of the whole pathovar. In addition, a lack of function in some of these strains was previously suggested for the raxST genes encoding a tyrosine sulfotransferase [90]. These observations suggest that the AvrXa21 function might not be present in this pathovar.

The set of type III effectors of Xam CIO151 is comparable in size with those of other xanthomonads

Effector proteins secreted by the T3SS play an important role in pathogenicity and virulence in Gram-negative bacteria. A total of twenty-eight CDS were identified as candidate effector genes (Table S8), based on homology searches against a database that includes type III effectors from different bacteria, including plant and animal pathogens (Rodríguez-R & Koebnik, unpublished data). Early stop codons were predicted for nine of these CDS, belonging to families XopAD, XopAG, XopF and XopP. In order to further confirm the pseudogenization of these fragments, we tested their expression in transcriptomic data of strains overexpressing the hrpX regulator (data not shown) using Bowtie aligner [91] followed by the Velvet assembler [92]. Analysis of transcriptomic data did not show any expression for xopP or xopAG, supporting their potential pseudogenization. In addition, the fragmentation of xopAG into two consecutive ORFs and the two adjacent copies of xopP were confirmed through comparisons with the sequences reported by Bart and collaborators [15]. The two copies of XopP are: one corresponding to the middle part of xopP (xanmn_chr10_0524) and the other copy fragmented in three consecutive ORFs (xanmn_chr10_0523, the N-terminal; xanmn_chr10_0522, the middle part; and xanmn_chr10_0521, the C-ter). We found expression of XopF in our transcriptome (data not shown) and the stop codon identified in the genome was confirmed. This suggests that the shorter version of xopF is still expressed.

The remaining nineteen genes (including xopF) belong to seventeen effector families ( Table 3 ). Several fragmented sequences were inferred as part of candidate TAL effectors (AvrBs3/PthA family). Based on Southern blot analyses, strain CIO151 has two TAL effectors [26]. However, assembly of the genomic regions that encode TAL effectors is not accurate due to the presence of quasi-identical repeats in the TAL effector genes. Therefore, it is challenging to accurately determine the number and sequences of candidate TAL effector encoding genes from short read, draft genome assemblies [15].

Table 3. Comparison of putative effector proteins from Xam CIO151 and other members of the Xanthomonas genus.

Effector family	XamCIO151	Xeua	XooMAFFa	Xaca	Xfa1b	XccATCCa
AvrBs1	-	1	-	-	-	1
AvrBs2	1	1	1	1	1	1
AvrBs3	2¤	-	17	4	2	-
XopB (HopD1)	-	1	-	-	2*	-
XopC1	-	1	-	-	-	-
XopC2	1+	2*	1	2*	-	-
XopD	-	1	-	-	-	1*
XopE1	1	1	-	1	1	-
XopE2	-	1	-	1	1	1
XopE3	-	-	-	1	1	-
XopE4	1	-	-	-	1	-
XopF1	1(F)	1	1	-	-	1*
XopF2	-	1	-	1*	1*	-
XopG	-	1	1*	-	-	1
XopH	-	1	-	-	-	1
XopI	-	1	1*	1	1	-
XopJ	-	2	-	-	1	1
XopK	1	1	1	1	1	1
XopL	1	1	1	1	1	1
XopN	1	1	1	1	1	1
XopO (HopK)	-	1	-	-	-	-
XopP	4* +	1	1	1	1	1
XopQ (HopQ1)	1	1	1	1	1	1
XopR	1	1	1	1	1	1
XopT	-	-	1	-	-	-
XopU	-	-	1	-	-	-
XopV	1	1	1	1	1	-
XopW	-	-	1	-	-	-
XopX	1	1	1	1	1	2
XopY	-	-	1	-	-	-
XopZ (HopAS1)	1	1	1	1	2*	1
XopAA	-	1	1	-	-	-
XopAB	-	-	1	-	-	-
XopAC	-	-	-	-	-	1
XopAD (SKWP)	3* +	3*	1	1	1	-
XopAE (HpaF)	1+	2*	1	1	1	-
XopAF (HopAF1)	-	-	-	-	1	-
XopAG (HopG1)	2*	-	-	-	1*	1
XopAH	-	-	-	-	-	1
XopAI	-	-	-	1	1	-
XopAJ	-	1	-	-	-	-
XopAK (HopK1)	1	1	-	1	1	-
XopAL	-	-	-	-	-	2
XopAM	-	-	-	-	-	1
XopAOc	2(+1)	-	-	-	-	-
Total&	19	27	37	22	22	21

Number in parentheses indicates number of genes in corresponding category.

^aData from effector families summarized by White et al. [13] and www.xanthomonas.org.

^bData reported by Moreira et al. [78].

^cNew effector family described by Potnis et al. [14].

^*Possible pseudogenes.

⁺Located in predicted genomic island.

⁽⁺¹⁾One copy located in predicted genomic island.

^FWith a premature stop codon but may be still functional.

^¤Incomplete sequence due to repeats.

^&Total number of potentially functional genes.

Because T3Es might define the host range and tissue specificity of xanthomonads, we compared the putative set of T3Es in Xam CIO151 with that of other members in the genus Xanthomonas ( Table 3 ). This approach revealed a comparable number of effector families between Xam CIO151 and Xeu, Xac, Xanthomonas fuscans subsp. aurantifolii str. ICPB 11122 (Xfa1) [78] and Xcc species, each species having a specific T3E repertoire. In addition, the number of effector families, which appear to be non-functional due to the presence of early stop codons or frameshifts, does not significantly differ between Xam CIO151 and other sequenced xanthomonads (data not shown).

Genomics studies of Xanthomonad species have revealed a core set of conserved T3Es. This set includes AvrBs2, XopK, XopL, XopN, XopQ, XopR, XopX and XopZ, and was confirmed in Xam CIO151. In order to study the evolution of this set of proteins inside the genus Xanthomonas, a phylogenetic reconstruction was performed using these conserved effectors. XopZ and XopX were excluded due to the presence of more than one copy in some of the species. Hpa1 (XopA), which is a conserved type III-secreted protein of Xanthomonas, was also included in this analysis ( Fig. 4 ). The phylogenetic tree suggests a common origin for the core effectors in the X. axonopodis clade consisting of Xeu, Xac, Xfa1 and Xam CIO151. Our results are consistent with a previously published genomics scale phylogeny of the genus [93], suggesting that the evolution of these genes resembles species relationships within the genus and that they were probably present in the common ancestor of these taxa. In addition, the degree of conservation of this set of proteins within the genus Xanthomonas and the lack of co-location with recent HGT events in Xam CIO151 suggests that the acquisition of this set of effectors appears to be an ancient event.

Figure 4. Phylogeny of conserved effectors in the genus Xanthomonas.

Phylogenetic tree of concatenated conserved effector protein sequences of AvrBs2, XopK, XopL, XopN, XopQ XopR families and the Hpa1 protein, obtained with a Bayesian approach. Numbers on branches indicate Bayesian support values. Length of branches indicates the number of amino acid substitutions per site.

Two genes (xanmn_chr06_5019 and xanmn_chr07_0047) showed high similarity, 95% and 81% at the amino acid level, to the recently reported effector family XopAO [14]. BLASTN and BLASTP searches in all available Xanthomonas genome sequences revealed that the XopAO effector family appears to be restricted to X. gardneri (Xg) and Xam CIO151, with Xam CIO151 possessing two members of this family. The Xg xopAE gene as well as one of the Xam xopAE genes are encoded in a region with atypical nucleotide composition ( Table 3 ), which supports the possible acquisition of these genes through horizontal transfer events.

Other determinants of interactions with plants in Xam CIO151

We also mined the genome sequence of Xam CIO151 for other pathogenicity-related determinants, such as chemotaxis, motility, the synthesis of second messengers and selected metabolic pathways, among others. This allowed us to generate a complete record of possible pathogenicity determinants. These analyses indicated the presence of genes related to chemotaxis, type IV pili (with corresponding genes distributed along the sequence of Xam CIO151), flagella (with a typical clustering of the corresponding genes), siderophore biosynthesis involved in iron uptake, and putative polyketide synthases ( Table 2 ).

We also identified candidate adhesins in the genome sequence of Xam CIO151. Twenty four genes which are highly similar (identity ≥95% on more than 90% of the protein length) to Xeu or Xac type IV pilus (T4p) genes were identified in Xam CIO151 genome. The other T4p genes identified in Xeu and Xac genomes were not detected in Xam CIO151 or were too distantly related. Although no gene coding a pilin was identified among these genes, the presence of the genes encoding the sensor protein PilS, the ATPases PilB and PilT, may indicate that this T4p could be functional in Xam CIO151. Concerning the non-fimbrial adhesins, potential orthologs of autotransporters and filamentous hemagglutinins were identified in Xam CIO151 genome. One gene (chr06_0342) identified as yapH2 and orthologous to XCV2103 harbors the specific domains of autotransporter adhesins. Two putative xadA genes were detected in Xam CIO151 genome (corresponding to locus tags chr_12_0029 and chr_12_0030 for the first one and to locus tags chr12_0032 and chr_12__0096 for the other). These putative xadA genes are split into two fragments in the genomic sequence. Regarding the fhaB family, Xam CIO151 has a gene similar to the filamentous hemagglutinin XAC4114, and two consecutive pseudogenes similar to the hemolysin activator protein XAC1815, which supports the duplication and decay observations from Mhedbi-Hajri and collaborators [94]. On the other hand, the fhaC gene, which is proposed as a helper in the translocation of FhaB adhesins, was not identified in Xam CIO151. The absence of this gene has also been reported in Xeu [94]. Due to this fact, the functionality of genes in the fhaB family is not clear in this Xam strain.

The role of cell wall degrading enzymes (CWDEs) Xam CIO151 pathogenicity has not been documented yet. However, they could be responsible for the ability of Xam to colonize xylem vessels, as it has recently been suggested for eukaryotic plant pathogens [95]. Based on homology analyses and manual genome annotation, we defined an inventory of 30 putative CWDEs and three related pseudogenes in Xam CIO151, including eight cellulases, two pectate lyases, five xylanases, two rhamnogalacturonases, one polygalacturonase, two endoglucanases, five beta-glucosidases and five alpha-glucuronidases (Table S9). All 30 candidate CWDEs have orthologous genes in other xanthomonads. Interestingly, the xylanase-encoding gene xynB seems to be absent, as observed for Xv, Xg and Xcc species [14]. Three consecutive cellullase genes were identified in a region with atypical composition. A gene encoding an alpha-glucuronidase of the GH67 family (agu67), which is present in all sequenced xanthomonads and which is clustered with xylanase genes in some xanthomonads, could be non-functional in Xam CIO151 due to a stop codon present in the middle of the coding sequence. This glucuronidase plays a joint role with xylanases during degradation of xylooligosaccharides in Pseudomonas cellulosa [96], and it has been proposed as a favorable element during the xylan degradation due to the limited ability of xylanases for cleaving uronic acid bones [97]. Hence, the absence of xynB suggests that xylan degradation is not highly efficient in Xam CIO151.

Using the genome of Xam CIO151 as a source for easily accessible genotyping tools

Genomic resources offer the unique opportunity to develop genome-based molecular typing tools, such as MLVA schemes [34]. We used the genome sequence of Xam strain CIO151 to predict and evaluate VNTR loci. To develop a robust typing scheme, candidate loci were predicted for Xam strain CIO151 and three additional, phylogenetically close Xanthomonas strains, X. axonopodis pv. citri strain 306, X. campestris pv. vesicatoria strain 85-10, and X. axonopodis pv. citrumelo strain F1, using a web-based pipeline. Predicted loci for Xam that were also present and apparently polymorphic among these strains were targeted for primer design based on conserved sequence stretches within the flanking regions. A total of 14 primer pairs were generated and tested by PCR on 14 strains representative of the worldwide diversity of Xam (Tables S10 and S11). High-resolution agarose gel electrophoresis and/or DNA sequencing revealed that all 14 loci are polymorphic among this set of strains ( Fig. 5 , and data not shown).

Figure 5. Molecular analysis of selected VNTR loci of Xam.

PCR amplicons of VNTR loci of Xam were separated by agarose gel electrophoresis. A, XaG1_02 (362 bp); B, XaG1_29 (251 bp); C, XaG1_58 (192 bp); D, XaG2_50 (119 bp); E, XaG1_12 (111 bp). For comparison, expected sizes for Xam strain CIO151 are given in brackets.

To increase the number of loci, we also developed PCR primers for eight additional VNTR loci that appeared to be specific to Xam (Table S10). Since 65 draft genome sequences became available to us, we tested all 22 predicted loci on this valuable set of strains representing over 11 countries of origin belonging to three continents, and 70 years of collection [15]. We could detect and extract corresponding VNTR loci from 39 (XaG1_70) to 65 strains (average: 59 strains) (Table S12). Multiple alignments allowed us to estimate the exact number of repeats for each locus and the number of different patterns (haplotypes) per VNTR locus ( Tables 4 and S12).

Table 4. Characteristics of VNTR loci for 65 Xam draft genome sequences.

VNTR locus	Repeat unit size1	Number of repeats		Haplotypes			Samples with incomplete
		Min.2	Max.3	Number of samples4	Number of haplotypes5	HGDI score6	stuttered repeats
XaG1_73	6	3	16	60	12	0.903
XaG1_67	6	9	22	51	12	0.900
XaG1_02	7	8	20	48	12	0.894
XaG1_29	7	10	22	48	10	0.854
XaG1_71	6	3	12	57	9	0.778	23%
XaG2_50	6	5	12	61	7	0.774
XaG1_70	7	10	15	39	6	0.773	8%
XaG1_72	6	4	11	59	8	0.710
XaG1_65	6	7	12	65	6	0.670
XaG1_58	6	3	9	65	7	0.658
XaG1_12	7	4	9	63	5	0.635	35%
XaG2_52	13	4	10	52	7	0.839
XaG2_37	24	1	2	64	2	0.476
XaG2_55	12	2	5	65	3	0.146
XaG1_105	8	3	11	64	8	0.694
XaG1_101	7	4	6	63	3	0.618	24%
XaG1_108	6	3	4	65	2	0.306
XaG1_110	21	2	4	59	3	0.523
XaG2_109	26	2	3	56	2	0.486
XaG2_106	22	2	3	65	2	0.170
XaG2_116	22	1	2	65	2	0.089
XaG2_117	25	1	2	64	2	0.031

¹: Repeat unit sizes are given in bp.

^{2 and 3}: Minimal and maximal numbers of repeats (only those in integer numbers) are given.

⁴Number of samples with a complete VNTR locus in the draft genome sequence is given.

⁵Number of different VNTR patterns (haplotypes) is given.

⁶Hunter-Gaston discriminatory index (HGDI) scores are given.

Discriminatory indices (HGDI) were calculated for all VNTR loci ( Table 4 ). HGDI scores varied significantly from 0.031 for XaG2_117 to 0.903 for XaG1_73. As described previously [98], VNTR loci were classified into highly (>0.6), moderately (0.3 to 0.6), and poorly (<0.3) discriminating based on the HGDI scores. Of the 22 loci, 14 loci showed high discriminatory power. Four loci, XaG1_108, XaG1_110, XaG2_37 and XaG2_109, were found to be moderately discriminatory, and four loci, XaG2_55, XaG2_106, XaG2_116 and XaG2_117, had only poor discrimination. Four loci, XaG1_12, XaG1_70, XaG1_71 and XaG1_101, contained incomplete stuttered repeats which would pose problems in high-throughput analyses using multiplex PCR and amplicon analyses via capillary electrophoresis, resulting in uncertain calls of repeat numbers. These less useful loci, as well as the two loci with extremely poor discrimination (XaG2_116 and XaG2_117), were not considered for a broadly applicable MLVA scheme. Strikingly, combining the remaining 16 VNTR loci into an MLVA-16 scheme allowed resolution of almost all strains. Altogether, at least 57 haplotypes were observed among 65 strains, corresponding to 49 singletons and 8 doublets. Seven of the eight doublets include up to four VNTR loci for which the number of repeats could not be estimated for at least one strain due to the draft status of the genome sequence. In these cases, any number of repeats was taken into account when grouping strains into haplotypes. Hence, it is possible that these seven doublets could be experimentally resolved by the MLVA-16 scheme. Three doublets (IBSBF2345/IBSBF2346, IBSBF2672/IBSBF2673, UG24/UG27) originated from the same country and have been isolated in the same year (Brazil 2006, Brazil 2009, Uganda 2011). Two doublets (Xam669/IBSBF320, IBSBF2666/IBSBF2820) originated from the same country and have been isolated in two successive years (Brazil 1973/1974, Brazil 2009/2010). Another doublet (CFBP1851/CIO151) corresponds to two isolates from Colombia from years 1974 and 1995. The seventh doublet (UA556/IBSBF2818) was isolated in Colombia in 2009 and in Brazil in 2010. For the last doublet (ORST4/NCPPB1159), the origin of one strain, NCPPB1159, is unknown [15]. These results indicate that most if not all doublets likely correspond to truly related strains and do not result from homoplasy at certain VNTR loci. The MLVA-16 scheme translates into an excellent HGDI score of 0.996, thus demonstrating its suitability for typing of Xam strains.

Discussion

This first expert-annotated high-quality draft genome sequence of Xam offers new insights into the genome structure of a bacterial plant pathogen affecting cassava crops and its candidate pathogenicity determinants. The comparison of the Xam CIO151 genome with other sequenced xanthomonads allowed us to propose a putative repertoire of 126 genes unique to Xam CIO151 that could play an important role in the cassava-Xam CIO151 interaction, possibly shaping properties such as host range and pathogenesis. Notably, among these genes, we identified both putative secreted proteins and membrane proteins, molecules widely recognized as bacterial pathogenicity determinants. Therefore, this group of genes constitutes an excellent candidate set for further research in order to understand the molecular basis underlying CBB and development of comprehensive control strategies for this disease.

The similarity of the Xam CIO151 genome sequence with those of Xeu and Xac, as well as the identification of clusters previously described as conserved in other xanthomonads, support the correct assembly of the genome sequence. Interestingly, in this and previous studies, phylogenomic relationships inferred from coding sequences (clusters of orthologous groups and conserved effectors) have placed Xam CIO151 within a monophyletic clade with Xac and Xeu, sister to the Xoo containing clade, and more distantly related to Xcc (Figure 3; [94]). Nonetheless, the similarity in the global genomic structure between Xam and that of other xanthomonads does not perfectly match such phylogenomic clustering. While the genomic structure of Xam CIO151 is more similar to that of Xcc and not to that of Xoo thus conflicting with the phylogenetic expectation This can be explained by the high number of recombination events reported in Xoo and the high number of insertion sequences [49], [99] Similar findings has been reported in the comparison between Xac and Xoo MAFF in global genome analysis [99]. These findings suggest that evolutionary processes leading to changes in genome organization are not entirely reflected in the conservation of genes.

Special features of Xam CIO151 such as a particular set of candidate T3Es and related pseudogenes were also identified. There were several T3Es present in Xam CIO151, which are not part of the conserved group among xanthomonads and which could be important determinants for its ability to colonize cassava. TAL effectors represent a challenge for second generation sequencing technologies at the stage of assembly of the central repeat region, and this was the case for Xam strain CIO151 as well. However, a member of this family has been demonstrated to be important for the ability of Xam to infect cassava [16]. The genome of Xam CIO151 encodes two TAL effectors and ongoing studies seek to determine their importance in the virulence of this strain.

As in other pathogenic bacteria, HGT events probably have contributed to the repertoire of T3Es in Xam. For instance, we detected XopAO in Xam CIO151, an effector that had only been identified in one more strain of xanthomonads, namely X. gardneri, where its functionality was confirmed and its origin from Pseudomonas via HGT was proposed [14]. Consistent with this speculation, our analyses support the hypothesis that Xam CIO151 acquired this effector via a recent HGT event. Additionally, we observed that the xopP pseudogenes and xopC2 gene were located in a putative PAI (regions with atypical composition, mobile sequences and next to tRNA genes), whereas putative members of the XopAD and XopAE effector families were encoded by regions with atypical nucleotide composition. Altogether these observations suggest an important role for HGT events in recruiting a specific pathogenicity arsenal with probable consequences for microbial adaptations.

Four families of effectors may be pseudogenes in the genome sequence of Xam CIO151 (XopAD, XopAG, XopF and XopP). These effectors also seem inactive or absent in some but not all strains of Xam sequenced to-date [15] suggesting that their presence may be dispensable when infecting cassava plants. Consequently, when these T3Es were screened in a larger sample of Xam genomes, there was no correlation between the presence of a full-length version of these effectors and the levels of virulence [15]. It is possible that full-length functional versions of these proteins would induce an Effector-Triggered Immunity (ETI) in certain cultivars of cassava. No gene-for-gene interaction has been reported for this pathosystem. However, the quantitative resistance observed in certain cultivars might be the result of one or several of such interactions [7].

A Quantitative Trait Locus (QTL) for resistance to CBB in cassava co-segregates with a gene encoding a receptor-like kinase homologous to Xa21 from rice, a transmembrane protein containing an intracellular kinase domain [7], [8]. In the rice-Xoo system, rice immunity to Xoo is probably triggered after the recognition by the host Xa21 receptor of a bacterial secreted molecule [100]. The rax (required for AvrXa21 activity) genes in Xoo encode for a sulfotransferase, a two-component system and a transmembrane transport system. Comparisons with the Xoo sequence revealed frameshifts in the genes for the sulfotransferase [15] and the ABC transporter, which are both required for the AvrXa21 function. This finding suggests that Xam does not produce and secrete the molecule recognized by the rice Xa21 protein. Interestingly, a homolog of Xa21 co-segregates with a QTL for resistance to certain strains of Xam [7]. It is possible that this protein recognizes a molecule analogous to that expressed and secreted by Xoo. However, our findings indicate that secretion of this molecule is probably functionally different from that performed by Xoo in the Xa21 system.

There are known variations in T2SS substrates among xanthomonads, even for conserved CWDEs in different species. For example, conserved substrates among several xanthomonads are apparently not secreted by the T2SSs in Xeu [101]. In addition, one of the T2SS clusters (xcs) is absent in Xoo [74] and, hence, the importance of this system for plant colonization is questionable. Taking this into account, future studies that evaluate the role of xps and xcs systems in Xam and their substrates are necessary to define their specific function, as well as to establish a curated set of CWDEs that are secreted via one or the other T2SS in Xam. In general, in terms of xylan degradation, the absence of the alpha-glucuronidase genes agu67 and xynB is suprising, since a xylan is a prominent cell wall component in cassava (14.3%; [102]) and one would expect cassava pathogens to express a range of enzymes for its degradation. On the other hand, it is possible that xynC, a validated substrate of xps in Xeu [101], is sufficient for xylan degradation during infection in cassava, since it appears to be functional. These findings motivate further studies to determine the sufficiency of xynC in xylan degradation by Xam and its implications in pathogenicity and virulence of Xam.

The closer examination of the xanthomonadin cluster in Xam CIO151 sought to understand the atypical white coloration of this organism. Strikingly, in Xam CIO151 most xanthomonadin biosynthesis genes seem to be functional and only one gene encoding an acyl carrier protein dehydratase, which is necessary for xanthomonadin synthesis in Xoo strains [59], appears to be disrupted. This observation suggests that this gene may be responsible for the phenotypic shift in colouration observed in Xam. Interestingly, a BLAST search of this gene against the genome of X. citri pv. mangiferaindicae gave negative results (data not shown), suggesting that this might also cause the white phenotype of this bacterium. As xanthomonadin pigments have been implicated in epiphytic survival in other xanthomonads [57], their absence in Xam would imply the evolution of additional or novel characteristics involved in the survival of this pathogen during the epiphytic and early colonization phases [57], [103].

We found unique patterns in the LPS cluster of Xam CIO151. The organization of the lipopolysaccharide (LPS) biosynthesis cluster is highly variable within the genus Xanthomonas [87]. Our findings show that the organization of Xam CIO151 LPS cluster is similar to that of Xac and XooBX08 suggesting that they may share a common ancestral LPS cluster. Simultaneously, the partial presence of LPS cluster in a region related to HGT is congruent with the hypothetical role of HGT as a source of diversity for this cluster, as has been proposed by Patil and Sonti [104].

Available genome sequences allowed us to develop and test a new molecular typing scheme. Sixteen out of twenty two tested VNTR loci were conserved in strains from three continents, Asia, Africa and South America and allowed to distinguish most of the Xam strains. Some of these loci might also be useful to type other, phylogenetically close pathovars of the same DNA-DNA hybridization group (group 9, [105]), including important pathogens of bean (X. axonopodis pv. phaseoli; group 9.4), alfalfae (X. axonopodis pv. alfalfae; group 9.2), soybean (X. axonopodis pv. glycines; group 9.5), lettuce (X. axonopodis pv. vitians; group 9.5), cotton (X. axonopodis pv. malvacearum; group 9.5), and cowpea (X. axonopodis pv. vignicola; group 9.6). The tremendous increase in whole genome sequencing will soon answer this question and lead to new, powerful typing tools for all economically important xanthomonads [6].

In conclusion, data mining based on the first annotated draft genome sequence of Xam CIO151 allowed, for the first time, the systematic cataloging of genes which might play a role in the interaction between Xam CIO151 and its host plant cassava, thus significantly increasing the sparse knowledge about molecular pathogenicity determinants of Xam. This new insight will pave the way for new approaches in the generation of durable resistant cassava varieties, thus leading to more efficient cassava disease management. New typing tools based on VNTR loci will allow an efficient and robust evaluation of population structures of Xam in different regions of the world and to implement comprehensive epidemiological surveillance, thus enabling better control of cassava bacterial blight.

Materials and Methods

Xanthomonas species abbreviation

Xanthomonas albilineans GPE PC7 (Xal)

Xanthomonas citri pv. citri str. 306 (Xac)

Xanthomonas campestris pv. campestris str. 8004 (Xcc8004)

Xanthomonas campestris pv. campestris str. ATCC 33913 (XccATCC)

Xanthomonas campestris pv. campestris str. B100 (XccB100)

Xanthomonas campestris pv. vesicatoria str. 85-10 (Xeu)

Xanthomonas fuscans subsp. aurantifolii str. ICPB 11122 (Xfa1)

Xanthomonas oryzae pv. oryzae KACC10331 (XooKACC)

Xanthomonas oryzae pv. oryzae PXO99^A (XooPXO99A)

Xanthomonas oryzae pv. oryzae MAFF 311018 (XooMAFF)

Xanthomonas oryzae pv. oryzicola BLS256 (XocBLS256)

Xanthomonas perforans 91–118 (Xp)

Bacterial strain and DNA sequencing

The partial genome sequence was obtained with 454 technology (shotgun and paired-end tags) at Eurofins MWG Operon in USA (http://www.eurofins.com). The sequencing process was performed on total DNA (including plasmidic material) from X. axonopodis pv. manihotis strain CIO151 [9] extracted and purified as previously reported [27]. This strain was deposited as CFBP7661 in the French Collection of Plant-Associated Bacteria (CFBP).

Genome assembly and structural and functional annotation

The genomic sequence of Xam CIO151 was assembled using Celera assembler [106]. A total of 40 scaffolds were produced. A manual inspection step allowed us to reduce the number of scaffolds to 36 by joining consecutive sequences, according to comparisons with Xeu, that have overlapping ends, or consecutive parts of conserved regions flanking rRNA operons. Total DNA that included possible extrachromosomal DNA was used in the sequencing process. For this reason, partial ordering of scaffolds was performed by alignments with the closely related genome sequence of X. euvesicatoria (Xeu) 85-10 (syn X. campestris pv. vesicatoria) [47]. This species was selected based on higher average nucleotide identity values [107] with Xam CIO151 [93]. Scaffolds that mapped to the Xeu genome were classified as the main chromosomal sequences (named xanmn_chr), scaffolds that mapped to plasmids in the genus Xanthomonas were classified as plasmidic (named xanmn_pla) and scaffolds that did not map against either of these two categories were classified as having an undertermined origin (named xanmn_unk). The circular representation of Xam CIO151 sequence was constructed using the Circos tool [108]. tRNAs search was performed using the tRNAscan-SE software [109]. Candidate rRNA operons were identified through a similarity analysis using sequences previously reported in other xanthomonads. The validation of the assembly was performed through an analysis of the GC content, genome length, search of genes already identified in Xam and comparisons to other Xanthomonas species using MUMmer 3 tool [110] and MAUVE [48].

The FrameD tool [111] was used for gene prediction because it increases the probability of identifying genes in sequences that might have problems with misread polynucleotides in some of the NGS technologies, including 454. It was trained with 100 conserved genes predicted with Glimmer3.02 [112]. Automatic and manual annotation was performed using iANT (integrated ANnotation Tool), as previously described [50]. The manual annotation was carried out by experts in subcategories of the Clusters of Orthologous Groups (COG) assigned by the automatic annotation. International experts on T3Es, adhesins, Two Component Systems, Mobile Elements, etc, collaborated in the manual annotation process. The manual annotation included addition and deletion of genes that were erroneously predicted by the automatic annotation system, labeling of genes that were splitted within scaffolds constructed with the Paired End data, correction of start codons taking into account homologous sequences and Ribosomal Binding Sites, specific assignation of names of genes where orthology had been positively tested. Identification of orthologous genes in other xanthomonads, protein domain descriptions, alignments and other characteristics were taken into account by annotators during the manual annotation process. The resulting information from assembly and annotation of the genome was deposited in Genbank as Bioproject number PRJNA202245 and in iANT at the website https://iant.toulouse.inra.fr/X.manihotis

Identification of unique elements of Xam CIO151

Proteins in Xam CIO151 that do not have homologs in other xanthomonads were identified by OrthoMCL using the default parameters [113] as reported previously [50]. Based on the assumption that fragmented sequences could be classified as unique elements due to length differences, we repeated a BLAST [51] search of the previously identified proteins in the set proteins of sequenced xanthomonads using an e-value threshold of 1e⁻¹⁰. Additionally, genes with indeterminate nucleotide positions or genes that codified proteins with length lower than 50 aminoacids were dismissed. A BLAST search in the GENBANK database was conducted for the remaining sequences.

Identification of putative horizontal transfer events

In order to identify regions with unusual composition, AlienHunter tool [114] was used. The predictions were evaluated though a search of tRNAs (including 5000 upstream and downstream), and mobile elements sequences available in the ACLAME database [115] (with an e-value threshold of 1e⁻⁵⁰), on the putative regions predicted by AlienHunter. Identification of genes in Xam CIO151 that are shared with other xanthomonads was performed using OrthoMCL. Insertion sequences in Xam CIO151 were defined using data derived from the annotation process.

Phylogenetic analysis

The phylogenetic reconstruction was performed using a concatenated sequence of conserved effector proteins: AvrBs2, XopK, XopL, XopN, XopQ, XopR and the Hpa1 regulon. Even though XopX and XopZ genes are conserved, they were excluded from this analysis because some species have multiple copies. Xanthomonads species included in the phylogenetic tree were: Xam CIO151, XccATCC, Xcc8004, XccB100, XocBLS256, XooKACC, XooPXO99A, XooMAFF, Xeu, Xac, Xp and Xfa1. A Bayesian approach using MrBayes software [116] with ten millions Markov Chain Monte Carlo, twenty percent of burnin, two runs and four chains, and a maximum likelihood approach using RaxML [117] with one thousand bootstrap replicates and an individual evolutionary model for each partition was used for the phylogenetic reconstruction. Protein alignments were produced with MUSCLE alignment tool [118]. Models of evolution were determined using the software Prottest [119], AIC criterion was used to determine the best-fitting model.

Identification of clusters and pathogenicity determinants

Identification of hrp, gum, rpf, xanthomonadin and secretion systems clusters was performed through a manual annotation using available information for these clusters in other xanthomonads. The G+C content was computed for some of these clusters using the window-acgt tool from the glimmer3.02 package [112]. Elements related to effector proteins, toxins, cell wall degrading enzymes, chemotaxis and motility were identified using annotation data. The list of candidate effectors in sequenced xanthomonads was obtained from the website described in [13].

Prediction of VNTR loci and design of PCR primers

The chromosomal sequences of Xam CIO151 and three phylogenetically close pathovars of Xanthomonas (X. axonopodis pv. citri strain 306 (GenBank accession number AE008923), X. campestris pv. vesicatoria strain 85-10 (GenBank accession number AM039952), and X. axonopodis pv. citrumelo strain F1 (GenBank accession number CP002914)) were scrutinized for the presence of the candidate VNTR loci using a web-based prediction pipeline (http://www.biopred.net/VNTR/; [25], [47], [120]. The Tandem Repeat Finder algorithm [121] was used and two sets of parameters were employed: (i) region length of 30 to 1000 bp, unit length of 5 to 9 bp, at least 6 copies and an identity of a least 80% between adjacent repeats, and (ii) region length of 20 to 1000 bp, unit length of 10 to 26 bp, at least 2 copies and an identity of a least 80% between adjacent repeats. Predicted VNTR loci were grouped according to their shared 500-bp flanking regions. Loci that were predicted for Xam and present in at least two other strains were further evaluated.

Homologous 500-bp regions next to the predicted VNTR loci were extracted from the chromosomal sequences and aligned using MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) [118]. PCR primers matching to conserved segments were designed using the Finnzymes website (http://www.finnzymes.fi/tm_determination.html; Table S10). Designed primer sequences were queried against the chromosomal sequences using the MFEprimer website (http://biocompute.bmi.ac.cn/MFEprimer/; [122]) to confirm that the primer pairs will only amplify one locus per genome.

PCR amplification, agarose gel electrophoresis and DNA sequencing

Candidate polymorphic loci were tested on a set of at least 14 strains of Xam representing worldwide diversity (Table S11). PCR amplifications were performed using genomic DNA of Xam strains as template DNA. Each PCR reaction was carried out in a final volume of 25 µl and contained 10–50 ng genomic DNA, 2.5 mM MgCl₂, 40 nM PCR primers, 2 mM dNTP and 1 unit of Taq DNA polymerase (Promega, USA). All reactions were run for 35 cycles, each consisting of 20 sec at 95°C, 30 sec at 52–58°C (depending on the primer pair), and 30–60 sec at 72°C, with an initial denaturation step of 3 min at 95°C and a final extension step of 10 min at 72°C. PCR products were separated on agarose gels and, if required, sent for custom DNA sequencing (Beckman Coulter Genomics, UK).

Computational analysis of VNTR loci among 65 Xam draft genome sequences

All VNTR primer pairs (Table S10) were also tested on 65 recently released draft genome sequences of Xam [15] using in-house developed scripts. Numbers of complete repeats were determined from multiple alignments of all draft genome sequences. The allelic profile of a given strain was defined as the repeat numbers at each VNTR locus. The discriminatory power of all VNTR loci were calculated using the Hunter-Gaston discriminatory index (HGDI), using the following formula:

where D is the numerical index of discriminatory power, N is the total number of strains in the typing scheme, s is the total number of different strain types, and xj is the number of strains belonging to the j^th type [123].

Supporting Information

Figure S1

Alignment of putative chromosomal scaffolds of Xam CIO151 and Xeu, Xac, Xcc8004 and XooPXO99A chromosomes using MAUVE software. A. Alignment between Xam CIO151 and Xeu, B. Alignment between Xam and Xac, C. Alignment between Xcc8004 and Xam CIO151, D. Alignment between XooPXO99A and Xam CIO151. Vertical red lines in Xam CIO151 indicate the scaffolds.

(TIFF)

Figure S2

Comparison of lipopolysaccharide gene clusters of Xam, XooBX08 and Xac. Homologous genes are represented by the same color. Dotted lines in Xam indicates the distribution of the cluster on two consecutives scaffolds.

(TIFF)

Table S1

Predicted proteins unique to Xam.

(DOC)

Table S2

Characteristics of chromosomal regions predicted with atypical nucleotide composition.

(DOC)

Table S3

Potential pathogenicity associated islands (PAIs) in Xam.

(DOC)

Table S4

rpf gene cluster of Xam CIO151.

(DOCX)

Table S5

Type II secretion systems in Xam CIO151.

(DOC)

Table S6

Type III secretion system (hrp cluster) in Xam CIO151.

(DOCX)

Table S7

Xanthan gum gene cluster in Xam CIO151.

(DOC)

Table S8

CDS of putative effector proteins detected in the genome of Xam CIO151.

(DOCX)

Table S9

Putative set of cell wall-degrading enzymes present in Xam CIO151.

(DOCX)

Table S10

Oligonucleotide primers, PCR conditions, and characteristics of VNTRs analyzed in this study.

(DOCX)

Table S11

List of Xam strains used to evaluate VNTR primers.

(DOCX)

Table S12

Presence and characteristics of VNTR markers in 65 genome sequences of Xam worldwide.

(PDF)

Funding Statement

This research was funded by a grant from the Colombian Administrative Department of Science and Technology (Colciencias, Contract 264-2008) (http://www.colciencias.gov.co/), from the Vicechancellors office for research at Universidad de los Andes and from the International Foundation of Science (C/4074-1) (http://investigaciones.uniandes.edu.co/index.php/en/) (http://www.ifs.se/). The Faculty of Sciences provided support for MLAO. Members (TB, SC, AD, PD, TDdB, LG, FG, PL, MAJ, EL, LN, SP, OP, IS, CV, VV, BS, RK) from French institutions acknowledge the support of the French Network on Xanthomonads and its funding agencies INRA, department SPE and CIRAD. Work in RK's laboratory is supported by grants from the French Agence National de la Recherche (ANR-2010-BLAN-1723, ANR-2010-GENM-013) (http://www.agence-nationale-recherche.fr/). LDN and TDDB were supported by French Agence National de la Recherche grants (Xopaque ANR-10-JCJC-1703-01 and TBDTomic ANR-08-BLAN-0193-01, respectively). Work at the LIPM is performed in the frame of the LABEX TULIP (ANR-10-LABX-41). LP is supported by the French Direction Générale de l'Armement (2011 60 091) (http://www.defense.gouv.fr/dga). VV is currently supported by a Marie Curie IOF Fellowship (EU Grant PIOF-GA-2009-235457) (http://ec.europa.eu/research/mariecurieactions/about-mca/actions/iof/) at Colorado State University (Bioagricultural Sciences and Plant Management). RB is supported by a NIFA (National Institute of Food and Agriculture) postdoctoral fellowship (Award 2011-67012-30662). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.