Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp. aurantifolii
- Leandro M Moreira1, 2,
- Nalvo F AlmeidaJr13,
- Neha Potnis3,
- Luciano A Digiampietri12, 14,
- Said S Adi13,
- Julio C Bortolossi5,
- Ana C da Silva9,
- Aline M da Silva2,
- Fabrício E de Moraes5,
- Julio C de Oliveira5, 6,
- Robson F de Souza2,
- Agda P Facincani5,
- André L Ferraz5,
- Maria I Ferro5,
- Luiz R Furlan7,
- Daniele F Gimenez5,
- Jeffrey B Jones3,
- Elliot W Kitajima10,
- Marcelo L Laia5, 8,
- Rui P LeiteJr17,
- Milton Y Nishiyama2,
- Julio Rodrigues Neto11,
- Letícia A Nociti5,
- David J Norman18,
- Eric H Ostroski14,
- Haroldo A PereiraJr5,
- Brian J Staskawicz19,
- Renata I Tezza5,
- Jesus A Ferro5,
- Boris A Vinatzer4 and
- João C Setubal15, 16Email author
© Moreira et al; licensee BioMed Central Ltd. 2010
Received: 16 December 2009
Accepted: 13 April 2010
Published: 13 April 2010
Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C.
We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein.
We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.
Citrus canker is a disease with worldwide distribution that has severe economic impact on the citrus industry [1, 2]. Disease symptoms consist of water soaked lesions that develop into blisters, then pustules, and, finally, cankers. In severe cases, citrus canker can lead to defoliation and premature fruit drop . Eradication of infected plants is the method of choice to control the disease where it is not yet endemic. When the disease is endemic, control is attempted by planting disease-free trees, limiting the spread between orchards, and using preventive copper sprays [4–6]. However, none of these measures controls citrus canker efficiently.
There are three types of citrus canker described in the literature: types A, B and C. Type A originated in Asia, probably in Southern China, Indonesia or India, and it is the type that is most widespread and causes the greatest economic damage [4, 7]. The other two types have only been found in South America. Type B (or false canker) was originally identified in Argentina in 1923. This type is present only in Argentina, Paraguay, and Uruguay , whereas type C is limited to the state of São Paulo, Brazil .
The causal agent of canker A is Xanthomonas citri subsp. citri (which we abbreviate as XAC for reasons explained below). XAC causes disease on many citrus species, with C. paradisi (grapefruit) and C. aurantifolia (Mexican lime) being most susceptible in the field and C. reticulata (mandarin/tangerine) and C. sinensis (sweet orange) being relatively tolerant [10, 11]. Importantly, no citrus species is resistant to XAC after artificial inoculation, suggesting that there is no true genetic resistance against XAC and that field tolerance is mainly due to variation in growth habit .
The genome of XAC strain 306 from Brazil was completely sequenced in 2002  and compared to the genomes of Xanthomonas species that are pathogenic in other plants [13, 14]. This comparative genomics approach has greatly accelerated the study of the molecular basis of pathogenicity and virulence of XAC. XAC has a hrp/hrc cluster coding for a type III secretion system (T3SS) that is used by the pathogen to inject virulence proteins, called effectors, into host cells. While several genes coding for putative effectors have been identified in the XAC genome, the single most important effector is PthA [3, 15]. Even in the absence of the pathogen, PthA induces canker-like symptoms when transiently expressed in plants . Deletion of pthA abolishes the ability of XAC to cause cankers . Intriguingly, PthA induces cankers in all citrus species while it triggers plant immunity in other plant species, thus being the prime determinant of XAC specificity toward citrus [16, 18, 19].
Two variant forms of canker A have been described. The first was found in Southeast Asia in 1998 infecting C. aurantifolia. The pathogen was classified as XAC variant A* . The second variant was isolated in 2003 in Southern Florida in C. aurantifolia and C. macrophyla (alemow), and was named Xanthomonas citri variant AW. AW strains have been shown to be a sub-group within A* . These strains are primarily pathogenic on C. aurantifolia and do not cause disease on C. paradisi, even after artificial inoculation [19, 20]. A T3SS effector, called AvrGf1, was found to contribute to the exclusion of C. paradisi from the A* host range . A recent study  suggests that A* strains (including AW) have a wider genetic diversity than the strains that cause A-type canker.
XAC, XauB and XauC have been compared phenotypically and analyzed phylogenetically. All three strains present polar flagella with perceptible motility when cultured in semi-solid media . They all grow in the presence of lactose, manitol, and celobiose. However, only XAC is able to grow in the presence of maltose and aspartic acid, and it is also capable of pectate and gel hydrolysis . XauB and XauC have little or no affinity for polyclonal antisera prepared against XAC, and XAC is susceptible to bacteriophages CP1 and CP2 while XauB and XauC are not . It is notable that XauB has fastidious growth in culture media where both XAC and XauC grow well, for example in Agar nutrient and tryptophan-sucrose-agar media. All three grow well in media rich in glutamic acid . Multilocus sequence typing and other molecular analyses [28–30] have shown that XauB and XauC are more closely related to each other than to XAC.
Under the rationale that the availability of the genome sequences and annotations of the causative agents of the B and C canker types can substantially improve our understanding of the genomic basis of the disease, we have sequenced the genomes of XauB and XauC to draft status. We have compared them with the genomes of XAC and other xanthomonads. Identified commonalities among the three canker genomes represent candidate genes that may help explain the differences between citrus canker and diseases caused by other xanthomonads. We have also identified numerous gene differences between the three citrus canker genomes. Some of these genes were previously shown to contribute to the virulence of XAC  and are thus primary candidates for explaining the higher virulence of XAC compared to XauB and XauC as well as the host range differences that exist between the three canker types.
A note on species abbreviations
The organisms studied in this work do not have names that are universally accepted. At the time when its genome was sequenced
the accepted name for XAC was Xanthomonas axonopodis subsp. citri. In the meantime, Xanthomonas citri subsp. citri has been validly published as a name of this organism [32, 33]. However, because the locus tag prefix of XAC genes is 'XAC' we opted for this abbreviation to avoid confusion along the text. Similarly, the causative agents for the B and C cankers are known by different names, but we use XauB and XauC as acronyms so that they are in agreement with their respective locus tag prefixes. For the B species we use the name X. fuscans subsp. aurantifolii type B and for the C species we use the name X. fuscans subsp. aurantifolii type C . We refer to the three organisms collectively as the citrus canker strains (abbreviated by CC strains/genomes).
Results and Discussion
General features of XAC, XauB, and XauC genomes.
with functional assignment
XAC strain 306 has two plasmids, pXAC33 (34 kbp) and pXAC64 (65 kbp). Based on similarities between contig sequences and XAC plasmid sequences it appears that both XauB and XauC have plasmids. At least 46% of plasmid pXAC33 sequence is found in XauB contigs (46% of plasmid pXAC33 sequence is found in XauC contigs); at least 61% of plasmid pXAC64 sequence is found in XauB contigs (55% for XauC). We do not have enough sequence data to ascertain the exact number of plasmids in each Xau genome.
At http://bioinfo.facom.ufms.br/xanthomonas we provide an interactive tool that allows user-defined gene content comparisons among all sequenced Xanthomonas genomes.
Genes shared by all three strains but not present in other Xanthomonadaceae species
XAC, XauB, and XauC have 65 families of orthologous genes specific to them when compared to all other fully sequenced members of the genera Xanthomonas and Xylella (Additional file 2: Table S2). Among these 65 families we have identified 11 syntenic blocks. Not surprisingly, almost half of these genes code for hypothetical proteins of unknown function. Of the genes with a predicted function the genes encoding the predicted effectors XopE3 (XAC3224) and XopAI (XAC3230) (discussed below) are especially noteworthy. The large number of genes coding for various kinds of transporters are also worth mentioning: four TonB dependent receptors including one with homology to the Escherichia coli receptor FepA, which is involved in transport of siderophores across the bacterial membrane  and seven ABC transporters, which might be used either for translocation of substrates from the citrus apoplast into the bacterial cell to provide nutrients for the pathogen, or, alternatively, for secretion of toxins (either bacterial toxins or expulsion of citrus metabolites toxic to the CC strains). An additional transporter specific to the three CC genomes (XAC3198) is an alkanesulfonate transporter substrate-binding subunit, which is reported to enable E. coli to use sulfonates other than taurine . Besides transporters, several genes encode metabolic enzymes (an amidase, an urea amidolyase, a peptidase, and a nitrilotriacetate monooxygenase). This conspicuous presence of transporters and metabolic enzymes suggests that the CC strains might have adapted to specific metabolites present in the citrus apoplast. However, this will need to be confirmed experimentally by comparing growth of wild type strains and strains mutated in CC-specific genes in apoplastic fluid of citrus species and of other plant species. One other interesting gene present in all CC genomes is a gene coding for a methyl parathion hydrolase (XAC0726), predicted to degrade the insecticide methyl parathion [38, 39]. Orthologs of this gene and other genes that degrade organophosphates are common in soil bacteria and in the soil-borne pathogen Ralstonia solanacearum but have not yet been found in any other foliar plant pathogen.
Some of these syntenic CC-specific regions are anomalous in terms of nucleotide composition as determined by the program AlienHunter  and may thus have been acquired by horizontal gene transfer.
The three CC genomes have important differences in regard to their repertoires of type III secreted effectors
The hrp/hrc genes encoding the T3SS are basically the same and found in the same order in all three CC genomes. However, there are notable differences in the three putative T3SS-secreted effector repertoires.
Putative effectors found in the XAC, XauB, and XauC genome sequences.
Pfam: functional/structural domain
Candidate effectors common to XAC, XauB, and XAUC
Glycerophosphoryl diester phosphodiesterase
AvrBs2 from X. campestris pv. vesicatoria 
XACa0039 (pthA2) XACb0015 (pthA3)
Transcriptional activator, nuclear localization
XopE1 (avrXacE1, hopX, avrPphE)
AvrXacE1, XopE1 from X. campestris pv. vesicatoria 
XopE3 (avrXacE2, hopX, avrPphE)
X. campestris pv. vesicatoria
Identified in Xoo by cya assay 
Inosine uridine nucleoside N-ribohydrolase
Identified in Xoo by cya assay 
Identified in Xoo by cya assay 
XopZ (HopAS, AWR)
XopAD (skwp, RSc3401)
SKWP repeat protein
Skwp from Ralstonia 
XopAI (HopO1 (HopPtoO, HopPtoS), HopAI1 (HolPtoAI))
XopAK (HopAK1 (HopPtoK, HolPtoAB)C terminal domain)
Not confirmed to be effector in Xanthomonas; homolog of effector in Pseudomonas
(associated with hrp cluster)
XAUC_20020 (associated with hrp cluster)
Pectate lyase, may not be T3SE
Candidate effectors present in XAC and XauB BUT ABSENT in XauC
XopE2 (avrXacE3, avrXccE1)
XopE2 found in another C strain 
Candidate effectors present in XauB and XauC BUT ABSENT from XAC
XopB (hopD1, avrPphD1)
New class introduced
C55-family cysteine protease or Ser/Thr acetyltransferase
XopAF (avrXv3, HopAF1 (HopPtoJ))
XopAG (AvrGf1, HopG1 (HopPtoG). HolPtoW)
Candidate effectors present only in XauC
As already mentioned, PthA is well known to be an important X. citri effector that plays an essential role in citrus canker, while limiting the host range of CC strains to citrus because it triggers immunity in all other tested plant species (see references above). The pthA gene is a member of the avrBS3 family of effector genes, members of which are present in most Xanthomonas genomes and in some R. solanacearum genomes . However, only PthA is known to induce citrus canker. Besides pthA (XACb0065), three paralogs of pthA are also present in the XAC genome (XACa0022, XACa0039, and XACb0015). All four copies are found on plasmids. The three paralogs do not seem to play an important role in citrus canker . We found two pthA homologs in the XauB genome (XAUB_40130 and XAUB_28490) and two in the XauC genome (XAUC_22430 and XAUC_24060/XAUC_09900 [the latter is a single gene with halves in different contigs]). Not all of these genes have been completely assembled due to the repetitive regions found in avrBS3 family members. However, El Yacoubi et al.  previously assembled a pthA homolog (pthB [GenBank: 2657482]) from the pXcB plasmid [GenBank: NC_005240] of a XauB strain with the same repeat copy number (i.e. 17.5) as pthA, and Al-Saadi et al.  sequenced and assembled another homolog (pthC [GenBank: EF473088]) from a XauC strain. These genes functionally complemented a pthA deletion in XAC without affecting host range . The XAUC_22430 gene has 99% nucleotide identity to pthC and thus probably corresponds to pthC and would be the functional pthA homolog of XauC. We do not have enough data to confidently report on the repeat copy number of the other three Xau pth homologs, but a phylogenetic analysis (see below) suggests that XAUB_28490 is the functional pthA homolog of XauB.
Effectors XopAI and XopE3 may play a role in citrus canker
A comparison of effectors present in all three CC strains with those present in fully sequenced Xanthomonas species, and data from the study by Hajri et al. , suggest that two additional putative effectors may play a special role in citrus canker. These are XopAI and XopE3. Both are present in all three CC genomes.
The putative effector xopAI is not found in any other sequenced Xanthomonas species and it was not included in the Hajri et al.  analysis. We do have evidence that it is present in Xanthomonas vesicatoria str. 1111 (Potnis et al., unpublished). Interestingly, the C-terminal region of XopAI has similarity to predicted ADP-ribosyl transferase domains of the effector HopO1-1 of Pseudomonas syringae and of hypothetical proteins in Acidovorax citrulli, Ralstonia solanacearum, and other bacteria. The N-terminus has high similarity to the N-terminus of the effector XopE2 of X. campestris pv. vesicatoria 85-10 as well the N-termini of a number of other Xanthomonas and Pseudomonas syringae effectors (more on the N-terminal region of xopAI below).
XopE3 belongs to the HopX/AvrPphE family of effectors. Effectors belonging to this family have been found in diverse phytopathogenic bacteria including Ralstonia, Pseudomonas, Acidovorax, and Xanthomonas, suggesting their conserved role in virulence on a wide range of hosts. Sequences from this family have similarity to the transglutaminase superfamily of enzymes, which are responsible for modification of host proteins . The HopX/AvrPphE effector from Pseudomonas syringae has been shown to be involved in host protein proteolysis, thereby suppressing host defenses [46, 47]. In xanthomonads, multiple effectors belonging to this group have been found, such as xopE1, xopE2, xopE3, xopE4. XopE1 and xopE2 have been found in most of the xanthomonads. XopE3 effector gene homologs have been found by PCR and dot-blot hybridization methods in some Xanthomonas axonopodis strains belonging to the alfalfae, anacardii, glycines, phaseoli, malvacearum, fuscans, mangiferae, indicae, and citrumelo pathovars . However, sequences of xopE3 from these strains could not be compared against homologs from CC strains since sequence data from the X. axonopodis strains mentioned are not currently available. Phylogenetic analysis of hopX orthologs shows that the xopE3 effector genes found in the CC strains group together with hopX1 effector genes from pseudomonads (data not shown).
Although all hopX orthologs show conservation of the catalytic triad (Cys, His, Asp residues) as well as the conserved domain "GRGN" N-terminal to the triad, the region C-terminal to the triad shows high degree of variability. This variable region has been hypothesized to be responsible for targeting different host proteins . In fact, while some AvrPphE (hopX) alleles from P. syringae pv. phaseolicola strains trigger gene for gene disease resistance in some bean cultivars, other alleles were shown to be virulent on these same cultivars. Amino acid differences in the C-terminal region of AvrPphE were identified between alleles . Similarly, comparing XopE3 homologs from different strains at the amino acid level and their corresponding reactions on different hosts might give clues regarding the variable C-terminal domains of XopE3 family members and might determine whether this variability is responsible for targeting different proteins in different host species.
Additional differences in effector repertoires among CC genomes
In addition to the pth differences noted above, other effectors that distinguish the XAC genome from the two Xau genomes are XopB, XopE4, XopJ (AvrXccB), XopAF (avrXv3), and XopAG, which are all present in both Xau genomes but absent from XAC strain 306. (AvrXccB homologs were found in two XAC strains by Hajri et al. .) The absence of these effectors from XAC strain 306 raises the possibility that these effectors might be responsible for limiting the host range of both B and C strains. Interestingly, XauB and XauC strains both contain xopAG, an effector gene belonging to the same effector family as avrGf1 from X. citri A w , which has been shown to be responsible for triggering a hypersensitive defense response in C. paradisi (grapefruit) . The xopAG gene from the B and C genomes shows 44% identity to avrGf1 at the amino acid level. The XauB and and XauC genes are almost identical to each other, with one important difference: in XauB xopAG is interrupted by a transposon. Therefore, the incompatibility between XauC and grapefruit and the ability of XauB to cause disease in grapefruit could be explained by this single gene difference. The xopE4 DNA sequence is identical in the two Xau genomes and has similarity to avrXacE3 but only with 31% identity at the amino acid level; this is why we named this gene xopE4 instead of xopE2. Unlike other XopE family members, XopE4 does not have a predicted myristoylation site, suggesting that it may not be targeted to the cell membrane as the other XopE family members.
Presence of an additional effector gene, the avirulence gene avrXccA2, has been shown in some X. aurantifolii B (CFBP3528, CFBP3530) and X. aurantifolii C (CFBP2866) strains by hybridization and PCR analysis . However, this avirulence gene was not found in the two sequenced Xau genomes. A homolog of the effector xopF1 (XAUC_20070) was found only in the XauC strain. It is located in a 5-kbp region that lies between the T3SS genes hrpW (XAUC_20020) and hpa3 (XAUC_20080). The same two genes are adjacent in XauB. Two transposases are present in this region, and the sequence of xopF1 has a frameshift, suggesting that this gene is likely the result of a recent insertion and is not active.
There are four effector genes present in the XAC and XauB genomes that have not been found in the genome of XauC: xopE2, xopN, xopP, and xopAE. These effectors could explain the wider host range of XAC and XauB compared to XauC, assuming a virulence activity of these effectors on citrus species. XopN has been shown to interact with the plant protein TARK1 and to interfere with immunity triggered by pathogen-associated molecular patterns (PAMP-triggered immunity) . Further experiments are required to determine the possible role of XopN in extending host range to lemon, grapefruit and sweet orange. Another effector that could have a similar role is XopAE (a hpaF/PopC homolog) [52, 53].
The harpin-like protein HrpW with a pectate lyase domain is present in all CC strains. In the sequenced XAC genome, it is not associated with the T3SS gene cluster, whereas in the genomes of XauB and XauC it is. The role of harpin-like proteins like HrpW as virulence factors or T3SS accessory proteins has not yet been determined in the Xanthomonas genus. Experiments will need to be performed to confirm translocation of the above putative effectors and their putative function as virulence or avirulence genes.
XAC-specific genes and genomic regions with respect to XauB and XauC
We have identified 25 groups of at least four consecutive genes that we term XAC-specific regions (XACSR) [Fig. 5, additional file 3 (Table S3), and additional file 4 (Fig. S4)]. Nearly all regions contain or are flanked by transposition elements or phage-related genes, suggesting that they could be the result of lateral transfer.
Several genomic differences are related to biofilm formation and quorum sensing
Xanthan gum is an exopolysaccharide that plays an important role in biofilm formation and hence in virulence of pathogens of the Xanthomonadaceae family [54–56]. Moreover, the synthesis of xanthan gum is regulated by variation in sugar concentration in the culture medium and by the activation of regulatory rpf genes [57, 58]. These genes are also responsible for the synthesis of diffusible signal factors, fundamental molecules for quorum sensing processes [59, 60]. Both Xau genomes contain an identical xanthan gum operon (XauC: XAUC_26940-27060; XauB: XAUB_007400-007410 and XAUB_10560-10450). The Xau genomes contain gene rpfH (XAUB_10500 and XAUC_27010), but this gene is not found in XAC. Gene rpfI, present in the xanthan gum operon of X. campestris pv. campestris strain ATCC33913, is absent from all three CC genomes.
Xanthan gum production.
2,57 (± 0,25)
3,93 (± 0,83)
1,70 (± 0,20)
1,62 (± 0,50)
2,35 (± 0,92)
1,82 (± 0,33)
In addition to the differences noted above, five additional XAC-specific regions (XACSR7, XACSR9, XACSR10, XACSR14 and XACSR17) may be related to its greater biofilm-formation capability when compared to XauB and XauC. Several of the genes in these regions facilitate adhesion in a process mediated by hemagglutinin .
XACSR7 contains two hemin storage system genes, hmsF and hmsH, and hemagglutinin coding genes (XAC1811-1816). Genes involved with acquisition and storage of hemin groups (hmsRFH) and type I secretion system genes (fhaC), and their secreted hemagglutinin (fhaB), are found in tandem and flanked by a tRNA R in the XAC genome (Fig. 6). In Yersinia pestis the hms genes are present in a cluster (the pgm cluster) related to temperature-dependent storage of hemin as well as expression of a number of other physiological characteristics . Mutations in these genes cause drastic decrease in Yersinia growth, preventing it from colonizing its point of entrance in infected flies (bucal orifice) [71, 72]. These genes also play a role in exopolysacharide synthesis, and reduction in biolfilm formation has also been observed in these mutants in Yersinia. Among all sequenced xanthomonads only XAC and X. oryzae have these genes. In both cases they are similar to (35 to 53% identity at the amino acid level) and syntenic with their homologs in Yersinia, E. coli K-12 and Erwinia carotovora (data not shown).
Recent work  describing mutations in the genes that code for hemagglutinin in Xylella fastidiosa strain Temecula (Pierce's disease) has shown that biofilm composition and virulence (adhesion and colonization) were affected in the mutants. This is consistent with results in XAC . This is evidence that the apparent absence of these genes in XauB and XauC might have the same effect (Fig. 6).
XACSR9 contains 19 genes. One of them (XAC1918) is a hemolysin-related gene. In enterobacteria hemolysins are an important virulence factor that are associated with proteins related to biofilm formation [75, 76]. The protein encoded by this particular gene interacts with VirD4, a Type IV secretion system component , which in turn may play a role in biofilm formation and cell aggregation, as observed for E. coli.
XACSR10 contains several noteworthy genes. Gene XAC2151 codes for the YapH protein. Its homolog (XOO2380, 84% identity at the amino acid level) in X. oryzae pv. oryzae KACC10331 when mutated drastically reduced the pathogen adhesion to plant tissue, thus decreasing its virulence ; in addition a homolog of this gene in X. fuscans subsp. fuscans CFBP4834-R (the causative agent of bacterial blight of bean, Phaseolus vulgaris), was required for adhesion to seed, leaves, and abiotic surfaces . XAC2197 and XAC2198 code for hemolysin-type calcium binding proteins, whereas XAC2201 and XAC2202 code for hemolysin secretion protein D (HlyD) and hemolysin secretion protein B (HlyB), respectively. The latter four genes do not have Xanthomonas matches in the sequence databases; they are similar instead to protein sequences from Acidovorax, Xylella and Pseudomonas species. The best hits are from Acidovorax avenae subsp. avenae ATCC19860, which is also a plant pathogen.
XACSR14 contains genes related to the type IV pilus-dependent system (Fig. 6). This system takes part in several processes, including adhesion, motility, microcolony formation, and protease secretion . In Xylella fastidiosa functional studies of these genes have shown that they are crucial for the host colonization process [82–86].
XACSR17 is also related to biofilm formation. Laia et al.  observed decrease in biofilm activity and virulence in four mutants with changes in this region (XAC3245-14G01/14G12, XAC3263-10G07/10G09, XAC3285-10F02 and XAC3294-17B04) (Fig. 6). The only mutated gene with functional assignment is rhsD (XAC3245). This gene has been described as coding for a membrane protein related to adhesion . In Xanthomonas campestris pv. campestris a RhsD protein has been found in the outer membrane vesicle associated with other virulence-associated proteins, such as HrpA/F/X/B4, HrcU, AvrBs1 and AvrBs2 . XAC contains a paralog of this gene (XAC2529), but it was not found in the Xau genomes either.
Gene wapA (XAC1305), not part of any XAC-specific region, and which is an adhesion facilitator by way of hemagglutinins , was not found in either of the Xau genomes.
XauB contains T4SS gene clusters similar to those found in Ralstonia solanacearum and in Agrobacterium tumefaciens
As reported by da Silva et al.  XAC has two T4SS clusters, one in the chromosome and the other in a plasmid (pXAC64). In XauB we found three clusters. Only one of them is similar to a XAC cluster (the pXAC64 cluster), and was therefore placed in group II, along with the T4SS cluster found on plasmid pXcB (already mentioned above in the context of the pth gene discussion). The other two XauB clusters were placed in groups I and III, respectively, while the XAC chromosome cluster was placed in group IV (Fig. 7A).
The XauB cluster placed in group III is similar to a cluster found in Ralstonia solanacearum, both in terms of organization as well as individual gene sequence similarity. This organization is quite different from those found in other bacterial species. This XauB cluster is found in a region containing 45 genes, all of which are XauB-specific when compared to other Xanthomonas genomes. Moreover this cluster is flanked by insertion elements. This evidence suggests that this cluster was likely acquired by lateral transfer.
The third XauB cluster was placed in group I, which contains T4SS clusters similar to those found in Agrobacterium tumefaciens C58 plasmid Ti , Xylella fastidiosa 9a5c plasmid pXF51 , and rhizosphere plasmids pIPO2T and pSB102 [94, 95]. This XauB cluster is most similar to those found in the rhizosphere plasmids, in particular to pIPO2T. The XauC draft genome sequence did allow the identification of one T4SS gene cluster, and it belongs to this group (Fig. 7A).
One of the members of this T4SS cluster is virD4 (XAC2623). Alegria et al.  have identified 12 XAC proteins that interact with VirD4. Six of these (XAC0096, 3266, 0151, 4264, 2609, 1918) are apparently absent from the Xau genomes, and five belong to XAC-specific regions (Fig. 8B). These genes might thus contribute to the increased virulence of XAC when compared to XauB and XauC.
Flagellum and motility
We did not find in XauB nor in XauC genes that lie between clusters F1 and F2; this region is XACSR9. In this region the gene XAC1927 has been shown to be important for citrus canker since mutations in it significantly decreased virulence  (Fig. 9A).
LPS and O-antigen genes
XACSR1 (XAC0037-0063) contains several genes related to LPS synthesis, including two copies of asnB (XAC0051 and XAC0059), which codes for an asparagine synthase. A homolog of this gene in Pseudomonas aeruginosa has been implicated in O-antigen biosynthesis .
The gene xacPNP
Gottig et al.  have identified a plant natriuretic peptide-like protein in XAC (xacPNP), encoded by gene XAC2654. They have shown that a XAC2654-deletion mutant resulted in more necrotic tissues and earlier bacterial cell death than in the wild type. XAC2654 lies between regions XACSR13 and XACSR14, and is flanked on both sides by phage-related genes. We have experimentally verified that neither XauB nor XauC appears to contain a homolog of XAC2654 (Additional file 5: Fig. S5). Therefore, xacPNP might be another gene contributing to the higher virulence displayed by XAC as compared to XauB and XauC.
Citrus canker continues to be an economically important disease. The publication of the XAC strain 306 genome in 2002 opened up new avenues of research, and several important insights into the genetics of canker have been obtained since then, most of them cited here. Yet, understanding the genomic basis of a bacterial plant disease is a complex undertaking. By obtaining the genome sequences of two additional citrus canker strains we have uncovered several new clues towards a thorough understanding of this disease.
We have approached the problem from two basic perspectives. The first was to determine commonalities among the three CC strains that were not found in other Xanthomonas genomes. Such traits are excellent candidates for the general genomic basis of canker and/or adaptation to citrus hosts. The second was to carefully compare the three CC genomes to one another, with special attention to genes that XAC has that the others (apparently) do not, as well as genes present in the Xau genomes but absent in XAC. Because of the draft nature of the Xau genomes here presented, all results concerning gene absence in their sequences are tentative. However, our hybridization platform provided additional evidence for the specificity of XAC genes and regions with respect to the other two strains.
Our most important findings are related to presence/absence of effector genes. In addition to the already known pthA gene, the genes xopE3 and xopAI deserve special attention in future studies. Moreover, we have identified several genes (such as xacPNP) that differentiate XAC from XauB and/or XauC. These genes or their homologs in other bacterial plant pathogens have demonstrated roles in virulence and/or host specificity. Hypotheses on their role in citrus canker and in host range differences between CC strains can now be tested experimentally.
We anticipate that knowledge in regard to CC-specific effectors and other CC-specific genes will be used in the future to engineer citrus species with durable resistance to citrus canker, thus reducing the economic impact of this disease on the citrus industry worldwide. Such knowledge will also be crucial for dealing with new canker variants that may emerge in the field, exemplified by the recent detection of what appears to be a new variant of Xanthomonas fuscans subsp. aurantifolii in swingle citrumelo .
Bacterial strains and DNA sequencing
The Xanthomonas fuscans subsp. aurantifolii type B genome sequenced was strain 11122 (B-69), isolated from a Citrus limon tree in Argentina. The Xanthomonas fuscans subsp. aurantifolii type C genome sequenced was strain 10535 (IBSBF338), isolated from a Mexican lime tree in São Paulo state in Brazil. We sequenced the genomes using the Sanger technique as described previously , with sequencers ABI 3700 and ABI 3100. For XauB we generated both shotgun and cosmid libraries; for XauC only shotgun libraries were created. For XauB we obtained 114,874 reads; for XauC we obtained 114,805 reads. We estimate this provided about 15× average coverage for each genome.
Microscopy and motility tests
Initial attempts to visualize the flagellum of the two Xau strains with a scanning electron microscope by the normal routine (adhesion of the cultured bacteria on a cover slip with the help of the cationic compound poly-L-lysine, fixation, dehydration and critical point drying) failed because apparently attachment of the flagellum is very weak and it tends to fall off easily. To circumvent the problem, a diluted suspension of the bacterial culture was directly transferred onto a cover slip and excess of liquid eliminated. A moist chamber was made in a Petri dish where the cover slip was inserted together with a small plastic vial containing about 1 ml of 2% aqueous osmium tetroxide. The Petri dish was sealed and wrapped with aluminium foil and left overnight. The next morning the cover slip was removed, air dried and sputter-coated with gold, mounted on the stub and examined in a LEO 435 VP scanning electron microscope. Results are shown in Fig. 9B.
For motility tests TSA and DYGS media were used . The Agar concentration of both media was changed (0.7%) so that the media were semisolid. For better growth visualization a phenol dye of 1% was added. Strains were placed in plates with solid media (TSA and DYGS) where isolated colonies were grown. The colonies were then placed in semisolid media. After 96 hours of incubation at 28°C, bacterial growth was observed in the test tubes, and results are given in the table in Fig. 9B.
Gum production determination
Strains were maintained both in autoclaved tap water at room temperature and at -80°C in NA medium (3 g/l meat extract and 5 g/l peptone) containing 25% glycerol. The three strains were picked from -80°C stock, streaked on solid TSA medium (10 g/l tryptone, 10 g/l sucrose, 1 g/l sodium glutamate, and 15 g/l agar) and grown overnight at 28°C. One single isolated colony from each strain was streaked again on solid TSA medium and grown overnight at 28°C. For XAC, a single colony was inoculated into 20 ml of liquid TSA medium (10 g/l tryptone, 10 g/l sucrose, 1 g/l sodium glutamate) in a 125 ml Erlenmeyer flask and incubated at 28°C in a rotary shaker at 180 rpm for 17 hours (1.100 OD at 600 nm). A 125 ml Erlenmeyer flask containing 50 ml of liquid TSA medium was inoculated with 1 ml of XAC culture and incubated at 28°C in a rotary shaker at 180 rpm for 5 hours (0.300 OD at 600 nm). This XAC culture was used as inoculum for xanthan gum production. For XauB and XauC, an inoculating loop was used to inoculate the bacteria from the solid TSA medium plates into 20 ml of liquid TSA medium in a 50 ml Falcon tube, followed by incubation at 28°C in a rotary shaker at 180 rpm for 24.5 hours (0.260 and 0.550 OD at 600 nm for XauB and XauC, respectively). One ml of XauB and XauC culture was inoculated into separate 125 ml Erlenmeyer flasks containing 50 ml of liquid TSA medium and incubated at 28°C in a rotary shaker at 180 rpm for 15 hours (0.300 OD at 600 nm). The XauB and XauC cultures were used as inoculum for xanthan gum production.
For gum production, 2.5 ml of each bacterial strain in liquid TSA medium (0.300 OD at 600 nm) was inoculated in three (triplicate) 250 ml Erlenmeyer flasks containing 100 ml of media for xanthan gum production (25 g/l glucose, 3 g/l yeast extract, 2 g/l K2HPO4, 0.1 g/l MgSO4.7H2O, pH 7.0 with 4 M HCl ) and incubated at 28°C in a rotary shaker at 178 rpm for 96 h. The cells from the 96 h culture were centrifuged at 9.666 g for 40 min. The bacterial pellets were stored at -20°C and the supernatants were transferred to 500 ml beakers. The gum was recovered from the supernatants by alcohol precipitation. Four grams of KCl were added to each beaker followed by agitation at room temperature for 15 min. Two volumes of cold isopropyl alcohol were added and the gum from each beaker was removed to pre-weighted plastic discs. After 16 h at 37°C the discs were weighted again and the gum amount was calculated. The bacterial pellet from each culture was also obtained. For this, each pellet was transferred to a pre-weighted beaker and weighted again after 14 h at 70°C.
From the shotgun libraries made for the sequencing of XAC strain 306 in 2001, 2,653 clones were selected for the design of a glass slide hybridization array. Inserts from selected clones were amplified by PCR with the M13-R and M13-F universal oligonucleotides. Products were purified and placed in duplicate slides, resulting in 6,144 probes, with 768 positive controls and 624 negative controls. Experimental validation was done by hybridization of total XAC DNA probes differentially stained, with 86% of all products with a hybridization signal greater than the average value for the noise signal plus two standard deviations. The final XACarray contains targets for the identification of 2,760 putative coding sequences (61% of all annotated protein-coding genes). The XACarray was used to compare XAC and XauC, and XAC and XauB, with XAC itself as a positive control. The results were similar (128 CDSs considered XAC-specific had similar ratios for both the XauB and XauC experiments, and 101 of these are in XACSRs); we report here detailed results only for the first experiment. Details of the array construction are described elsewhere (Moreira LM, Laia ML, de Souza RF, Zaini PA, da Silva ACR, Ferro JA, da Silva AM: Development and validation of a Xanthomonas citri subsp. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome, submitted).
Evidence for the absence of genes coding for XacPNP homologs in XauB and XauC was obtained by PCR using gene-specific primers (Forward: GGACCAACAACGAATATC; Reverse: ATGGGAATAGTCATGAAAC). XAC was used as positive control.
Assembly and genome annotation
Base calling, genome assembly and visualization were done with the phred-phrap-consed package [99–101]. Contigs were trimmed to remove low phred quality regions at both ends. For both XauB and XauC all contigs larger than 1 kbp have average phred quality greater than 20, and nearly all (99% for XauB and 96% for XauC) these contigs have average phred quality greater than or equal to 40 (i.e. accuracy equal to or better than 1 error in 10,000 bp). To improve assembly many transposase sequences were masked, and reads were re-assembled to obtain the final result. We estimated the fraction obtained of the complete genome of XauB by dividing the total length of contigs by the total genome size of XAC, and by adding 1% because of the removed transposases. We did the same with XauC. To estimate the average gap size we divided the number of contigs by the estimated size of un-sequenced genome.
Using paired-end reads scaffolds for the XauB and XauC chromosomes were obtained. The overall correctness of each scaffold was validated using a sliding-window GC-skew computation. Each scaffold was aligned against the XAC chromosome using MUMmer . Results are presented in additional file 1 (Fig. S1).
For determination of XAC-specific regions we relied on published data about putative genomic islands [14, 31, 105] and on AlienHunter results . Islands whose genes were not found in the XauB and XauC genomes by BLAST  analysis and with differential hybridization signals in the XACarray were considered XAC-specific regions.
We used a supermatrix approach as in previous work . Protein sequences of eleven Xanthomonas genomes (ingroup) and four Xylella genomes (outgroup) were clustered in 6,375 families using OrthoMCL . We then selected families with one and only one representative from each of the ingroup genomes and at least one outgroup protein, resulting in 1,666 families. Their sequences were aligned using MUSCLE  and the resulting alignments were concatenated. Non-informative columns were removed using Gblocks , resulting in 596,246 positions. RAxML  with the PROTGAMMAWAGF model was used to build the final tree.
The same methodology as above was used, with the following differences. Representative pth nucleotide sequences from Xanthomonas species were retrieved from GenBank, and added to the set of XauB and XauC pth nucleotide sequences. A pth gene from Ralstonia solanacearum [GenBank: CAD1557.1] was used as an outgroup in a preliminary round of tree construction to ascertain root position. The list of gene sequences used to build the tree is given in additional file 6 (Table S6). The Tandem Repeat Finder program  with parameters 2,7,7,80,10,50,500,1 was used to mask the internal repeats. The masked regions were removed and the resulting sequences were aligned with MUSCLE. A few manual adjustments to the multiple alignment were made before running Gblocks, which yielded 1,679 positions. RAxML with the GTRGAMMA model was used to build the final tree. The bootstrap values obtained for the clade indicated by an asterisk in Fig. 3 are given in additional file 7 (Fig. S7).
The candidate T3SS effectors in the XauB and XauC genomes were identified using tBLASTn  analysis and Pfam domain  searches. For tBLASTn analysis, all known plant and animal pathogen effectors were used as query with an e-value threshold ≤ 0.00001. Pfam domains were searched for possible domains found in known effectors in the predicted set of ORFs of draft genome sequences. Candidate effectors were classified according to the nomenclature and classification scheme for effectors in xanthomonads recently described by White et al. .
The draft genome sequences of XauB and XauC are available at GenBank under accession numbers ACPX00000000 and ACPY00000000, respectively.
Open Reading Frame
polymerase chain reaction
Type III secretion system
Type IV secretion system
Xanthomonas citri subsp. citri
Xanthomonas fuscans subsp. aurantifolii
Xanthomonas fuscans subsp. aurantifolii strain B
Xanthomonas fuscans subsp. aurantifolii strain C.
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), by Conselho Nacional de Desenvolvimento CientÌfico e Tecnológico (CNPq), by Coordenação para Aperfeiçoamento de Pessoal de Ensino Superior (CAPES), and by FUNDECITRUS, all of them Brazilian funding agencies. We thank the following students for their help: Aloisio J. Almeida Jr., Maria L. R. Carrera, Gustavo G. L. Costa, Daniel M. de Faria, Daniel P. Gasparotto, Rafael A. Homem, Vanessa C. Morgan, Julia M. Perdigueiro, Marcelo C. Perez, and Paula F. S. Tabatini.
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