Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris
Re e2VHt0oea0luls7.meea8r,cIshsue 10, Article R218 Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris
Yong-Qiang He 1
Liang Zhang 0
Bo-Le Jiang 1
Zheng-Chun Zhang 1
Rong-Qi Xu 1
Dong-Jie Tang 1
Jing Qin 1
Wei Jiang 1
Xia Zhang 1
Jie Liao 1
Jin-Ru Cao 1
Sui-Sheng Zhang 1
Mei-Liang Wei 1
Xiao-Xia Liang 1
Guang- Tao Lu 1
Jia-Xun Feng 1
Baoshan Chen 1
Jing Cheng 0
Ji-Liang Tang 1
0 CapitalBio Corporation , Life Science Parkway, Changping District, Beijing 102206 , People's Republic of China
1 Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, and College of Life Science and Technology, Guangxi University , Daxue Road, Nanning, Guangxi 530004 , People's Republic of China
Background: Xanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease of crucifers worldwide. The molecular genetic diversity and host specificity of Xcc are poorly understood. Results: We constructed a microarray based on the complete genome sequence of Xcc strain 8004 and investigated the genetic diversity and host specificity of Xcc by array-based comparative genome hybridization analyses of 18 virulent strains. The results demonstrate that a genetic core comprising 3,405 of the 4,186 coding sequences (CDSs) spotted on the array are conserved and a flexible gene pool with 730 CDSs is absent/highly divergent (AHD). The results also revealed that 258 of the 304 proved/presumed pathogenicity genes are conserved and 46 are AHD. The conserved pathogenicity genes include mainly the genes involved in type I, II and III secretion systems, the quorum sensing system, extracellular enzymes and polysaccharide production, as well as many other proved pathogenicity genes, while the AHD CDSs contain the genes encoding type IV secretion system (T4SS) and type III-effectors. A Xcc T4SS-deletion mutant displayed the same virulence as wild type. Furthermore, three avirulence genes (avrXccC, avrXccE1 and avrBs1) were identified. avrXccC and avrXccE1 conferred avirulence on the hosts mustard cultivar Guangtou and Chinese cabbage cultivar Zhongbai-83, respectively, and avrBs1 conferred hypersensitive response on the nonhost pepper ECW10R. Conclusion: About 80% of the Xcc CDSs, including 258 proved/presumed pathogenicity genes, is conserved in different strains. Xcc T4SS is not involved in pathogenicity. An efficient strategy to identify avr genes determining host specificity from the AHD genes was developed.
Xanthomonas campestris pathovar campestris (Xcc) is the
causal agent of black rot disease, one of the most destructive
diseases of cruciferous plants worldwide . This pathogen
infects almost all the members of the crucifer family
(Brassicaceae), including important vegetables such as broccoli,
cabbage, cauliflower, mustard, radish, and the major oil crop
rape, as well as the model plant Arabidopsis thaliana. Since
the late 1980s, black rot disease has become more prevalent
and caused severe losses in vegetable and edible oil
production in China [2,3], Nepal , Russia , Tanzania , and
the United Kingdom .
It has been shown that Xcc is composed of genetically,
serologically and pathogenically diverse groups of strains [4,8,9].
Certain Xcc strains are able to cause disease only in certain
host plants, indicating that there are incompatible
interactions between Xcc strains and their host plants. Flor's
genefor-gene theory  suggested that such an incompatible
interaction between microbial pathogens and plants
determines the pathogens' host specificity and is governed by an
avirulence (avr) gene of a pathogen and the cognate
resistance (R) gene of a host. Since the early 1980s, Xcc has been
used as a model organism for studying plant-pathogen
interactions [11-14] and more than one hundred Xcc
pathogenicity-related genes have been identified [13,15-19]. However,
few avr genes have been functionally characterized from Xcc.
Recently, whole genome sequences of two Xcc strains,
ATCC33913  and 8004 , have been determined.
Genome annotation predicted that Xcc possesses at least
eight genes that show sequence homology to the known avr
genes discovered from other bacteria [20,21]. Mutagenesis
analysis of these eight avr-homologous genes detected
avirulence activity for only avrXccFM .
Comparison of the whole genome sequences of the strains
8004 and ATCC33913 has revealed that the two genomes are
highly conserved with respect to gene content [20,21]. There
are only 72,521 bp and 5 protein-coding sequences (CDSs)
different between their genomic sizes and their total
predicted CDSs, respectively [20,21]. Although 170
strain-specific CDSs (108 specific for strain 8004 and 62 for strain
ATCC33913) were identified and three of the 8004
strainspecific CDSs were found to be involved in virulence [20,21],
the genetic basis about the host specificity of Xcc remains
unclear. As both strains 8004 and ATCC33913 were isolated
from the UK [20,21], they might be closely related strains
sharing a late common ancestor and this small genetic
variability might not represent the nature of Xcc genetic diversity.
To further determine the genetic variability and host
specificity of Xcc, in this work we collected 18 Xcc virulent strains
isolated from different host plants and different geographical
areas from North China to South China and compared their
genomes with the sequences of strain 8004 by array-based
comparative genome hybridization (aCGH).
The aCGH analysis has been used to study bacterial
pathogenicity, genetic diversity and evolution [23-31]. This
approach facilitates the comparison of un-sequenced
bacterial genomes with a sequenced reference genome of a related
strain or species. Genes in the organisms under study are
categorized into 'present' and 'absent/divergent' categories
based on the level of hybridization signal. The resolution
threshold of the aCGH is generally at the single gene level
(gene-specific microarray) , which is just appropriate for
identifying the genetic determinants responsible for host
specificity of plant pathogens that follow the gene-for-gene
relationship. This genomotyping technique has been used to
analyze phytopathogenic bacterial strain variation in Xylella
fastidiosa [33,34] and Ralstonia solanacearum .
In this paper we report the identification of a common
genome backbone and a flexible gene pool of Xcc revealed by
aCGH analysis. We also demonstrate that the type IV
secretion system (T4SS), which has been shown or proposed to be
involved in virulence of several bacterial pathogens [36-40],
is not engaged in the virulence of Xcc. Furthermore, three avr
genes were identified from the flexible gene pool by analysis
of the correlations between the occurrence of genes and the
reaction of different strains in different hosts followed by
experimental functional confirmation.
Characterization of Chinese isolates as Xcc
Twenty-two different strains/isolates were collected for this
study. Of these, the Xcc strain ATCC33913 is a type strain,
isolated from Brussels sprout (Brassica oleracea var.
gemmifera) in the UK in 1957 , and the Xcc strain 8004 is a
laboratory strain with spontaneous rifampicin-resistance,
derived from Xcc NCPPB No.1145 isolated from cauliflower
(B. oleracea var. botrytis) in the UK in 1958 . The other
20 isolates were collected from different infected cruciferous
plants in various geographic locations over a wide range of
latitudes across China and named CN01 to CN20 (Table 1).
These isolates were validated by morphological, virulent and
molecular analyses. All the isolates formed typical X.
campestris colonies of yellow mucoid texture on NYG agar medium
 and caused typical black rot disease symptoms on the
host plant radish (Raphanus sativus var. radicula; data not
shown). To further confirm the isolates, their partial 16S-23S
rDNA intergenic spacer (ITS) regions  were examined by
PCR and sequencing. A PCR fragment 464 bp in length was
obtained for every isolate except CN13 and CN19, for which
no PCR product was obtained. Sequencing results showed
that five isolates have identical ITS sequences to that of strain
8004, while the ITS sequences of the other 13 isolates differ
from that of 8004 by only one or two nucleotides (Additional
data files 1 and 2). The isolates CN13 and CN19 were not used
for further study in this work as they were not confirmed to be
Xcc by the 16S-23S rDNA ITS analysis. The phylogenetic
analysis by the maximal parsimony method  showed that
The origin of the Xcc strains used in this study
Host of origin
Cauliflower (Brassica oleracea var. botrytis)
Brussels sprout (B. oleracea var. gemmifera)
Chinese cabbage (B. rapa subsp. pekinensis)
Chinese cabbage (B. rapa subsp. pekinensis)
Chinese cabbage (B. rapa subsp. pekinensis)
Oilseed rape (B. napus ssp. oleifera)
Chinese cabbage (B. rapa subsp. pekinensis)
Chinese cabbage (B. rapa subsp. pekinensis)
Chinese cabbage (B. rapa subsp. pekinensis)
Radish (Raphanus sativus var. longipinnatus)
Chinese cabbage (B. rapa subsp. pekinensis)
Chinese cabbage (B. rapa subsp. pekinensis)
Cabbage (B. oleracea var. capitata)
Oilseed rape (B. napus subsp. oleifera)
Leaf mustard (B. juncea var. foliosa)
Chinese cabbage (B. rapa subsp. chinensis)
Chinese cabbage (B. rapa subsp. pekinensis)
Chinese cabbage (B. rapa subsp. chinensis)
Leaf mustard (B. juncea var. foliosa)
Chinese kale (B. oleracea var. alboglabra)
*The geographic coordinates of the Xcc strains in parentheses are estimated from information originating in the National Collection of Plant
the 18 proven Xcc isolates were grouped into two clusters and
each cluster contains previously identified Xcc strains
(Additional data file 2). These two groups were significantly
distinguished from other Xanthomonas species and X. campestris
pathovars (Additional data file 2), further confirming the 18
isolates as Xcc at the molecular level. The word 'strain' will be
used for the identified Xcc 'isolates' hereafter.
The virulence and hypersensitive response of Xcc
strains on different plants
The in planta pathogenicity test of Xcc strains was carried out
by the leaf-clipping inoculation method on eleven different
cultivars (cv.) of four cruciferous species (see Materials and
methods). The results showed that seven of the eleven
cultivars were susceptible to all of the Xcc strains tested,
whereas the other four plants manifested resistance to
particular Xcc strains (Tables 1 and 2). Based upon these results, a
gene-for-gene relationship governing the outcome of the
interactions between the Xcc strains and the host plants could
be postulated (Table 3). The key essentials are: first, the host
plants that were susceptible to all of the Xcc strains possess
no resistance genes against the Xcc strains; second, mustard
cv. Guangtou possesses a resistance (R) gene, arbitrarily
designated Rc1, for which the postulated interacting avirulence
(avr) gene is designated avrRc1, present in strains 8004,
ATCC33913, CN03, CN07, CN09, CN10, CN11, and CN20;
third, cabbage cv. Jingfeng-1 and radish cv. Huaye possess an
R gene named Rc2 that interacts with an avr gene named
avrRc2, present in strains ATCC33913, CN03, CN14, CN15,
and CN16; and fourth, Chinese cabbage cv. Zhongbai-83
possesses an R gene, Rc3, that interacts with the postulated
avrRc3 in strains 8004, ATCC33913, CN02, CN03, CN06,
CN07, CN08, CN12, CN14, CN15, CN16, CN18, as well as
CN20 (Tables 2 and 3).
We also examined the hypersensitive response (HR)  of
the Xcc strains on the nonhost pepper ECW10R, a plant
commonly used to test the HR of Xcc. The results showed that
eight hours after inoculation strains 8004, ATCC33913,
CN01, CN03, CN09, CN10, CN11, and CN20 elicited a typical
HR while the others did not (Table 2). According to the
results, we postulated that strains 8004, ATCC33913, CN01,
CN03, CN09, CN10, CN11, and CN20 possess an avirulence
gene, designated avrRp1, that interacts with a cognate
resistance gene, named Rp1, in the non-host plant pepper ECW10R
(Tables 2 and 3).
Sensitivity of aCGH analysis
To investigate genetic similarity and diversity among Xcc
strains, a DNA microarray encompassing 4,186 CDSs was
The plant assay results of Xcc strains
constructed, representing all CDSs (non-redundant) in the
reference strain 8004 . Primer design was based on the
genomic sequence of 8004, which is composed of 4,273 CDSs
. Of the 4,186 CDSs, gel electrophoresis revealed
successful amplification of 4,043 CDSs, representing 96.58% of the
non-redundant genome content. For the CDSs predicted to be
less than 100 bp in length, for which optimized primers could
not be designed, and those for which PCR amplification did
not work, a 70-mer oligo probe for each CDS was designed.
The word 'gene' will be used in reference to the CDS that each
spot corresponds to unless otherwise indicated.
To determine the sensitivity of our aCGH analysis, self-to-self
hybridization was performed using genomic DNA of the
reference strain 8004. After removal of faint spots for which the
intensity was lower than the average plus two standard
deviations of the negative controls (blank spotting solution) on
the array, it was found that more than 95% of all genes on the
array could be detected and the intensity ratio of the detected
genes lay between 0.6 and 1.6. aCGH analyses were then
carried out using the reference strain 8004 and its derivative
strain C1430nk, described previously . The strain
C1430nk is derived from 8004 and harbors the cosmid
pLAFR6 containing the open reading frames (ORFs) XC1429
and XC1430. The aCGH results revealed that only two genes,
XC1429 and XC1430, had an intensity ratio of approximately
1.9-2.4 (C1430nk/8004), indicating that sole copy alteration
at the genomic scale could be detected in this study (Figure 1).
Based on the above results, it was presumed that the
microarray can detect the 1.6-fold alteration when ignoring
sequence diversity. After passing the initial tests, aCGH
analyses were performed using the fully sequenced Xcc strains
8004 and ATCC33913. The results showed a good agreement
with the complete genome sequences of 8004 and
ATCC33913 (Figure 1). It was found that for the genes of
strain ATCC33913, whose sequences are >90% identical to
those of strain 8004, 99% of their spots on the array showed
intensity ratios 0.5. Therefore, intensity ratios 0.5 were
selected to be the threshold for genes detected as present/
conserved within strain 8004. Furthermore, 98% of the genes
previously reported to be specific to strain 8004 (that is, that
are absent in the genome of strain ATCC33913) were detected
Postulated avirulence genes in Xcc strains tested
as absent genes in the aCGH analysis of strain ATCC33913
(Figure 1). Our selected threshold for conserved genes here is
similar to that described by Taboada et al. , who used a
Log2 ratio (sample/reference) threshold of -0.8 to detect
conserved genes in aCGH analyses with an acceptable level of
type III secretion systems (T1SS-T3SS) as well as
extracellular polysaccharide production, and the rpf
(regulation of pathogenicity factors) gene cluster [11,12] are highly
conserved among the Xcc strains investigated, although some
predicted pathogenicity- and adaptation-related genes are
AHD (Table 5).
The validity of the aCGH results was further tested by PCR
examination of the presence or absence of 30 genes showing
a range of ratios in the aCGH analysis. The PCR primers used
and PCR results are presented in Additional data file 3. The
results show that a ratio (sample/8004 strain) of <0.5 gives
high confidence (98%) that the gene is absent/highly
divergent (AHD) in the sample strain.
Overview of the aCGH analyses of different Xcc strains
Using the parameters established above, the gene
composition of 18 Chinese Xcc strains was analyzed by aCGH using
the genome of strain 8004 as the reference. The results are
shown in Tables 4 and 5, Figure 2 and Additional data file 4.
Of the 4,186 CDSs spotted on the microarray slides, 3,405 are
conserved in all of the strains tested (Table 5). These
conserved CDSs may represent the common backbone ('core'
genes) of the Xcc genome, which contains most of the genes
encoding essential metabolic, biosynthetic, cellular, and
regulatory functions (Table 5). The genes relevant to central
intermediary metabolism, replication, transcription,
translation, the TCA cycle, and nucleotide, fatty acid and
phospholipid metabolism are largely conserved. Genes encoding the
components involved in the type I (T1SS), type II (T2SS) and
The aCGH results showed that 730 CDSs are absent or highly
divergent among all the Chinese strains tested (Tables 4 and
5). In addition, a total of 51 invalid hybridization spots (CDSs)
were observed in all the aCGH analyses of the 18 Chinese
strains. The 730 AHD genes, which account for 17.6% of all
valid hybridized CDSs in the aCGH analyses, may constitute
the Xcc flexible gene pool. The functional categories of all the
AHD genes are given in Table 5. Half of the AHD genes have
been predicted to encode proteins with unknown function.
The differences in the numbers of the AHD genes in different
strains are significant (Table 4). Compared with the reference
strain 8004, the most divergent Chinese Xcc strain is CN14,
of which 475 CDSs are AHD; and the most closely related
strain is CN07, of which only 137 CDSs are AHD. Fifty-seven
Xcc 8004 CDSs, most of them encoding hypothetical
proteins, are AHD in all eighteen Chinese strains. Of the 57
CDSs, 16 are conserved in strain ATCC33913. A hierarchical
clustering program  was used to explore the relationship
of the different Xcc strains based on the aCGH analysis
(Figure 2). The result shows that the Chinese strains and the
reference strain are divided into five groups (Figure 2). Some
Xcc strains classified in the same phylogenetic group based
10000 20000 30000 40000 50000 60000 70000
SFeignusirtievit1y determination of aCGH analyses
Sensitivity determination of aCGH analyses. (a) aCGH analyses of the
reference strain 8004 and its derivative strain C1430nk. The strain C1430
possesses one extra DNA copy of the ORFs XC1429 and XC1430
compared to the reference strain 8004. (b) TreeView display of the
aCGH clustering result of the two sequenced genomes of the Xcc strains
8004 and ATCC33913. Each row corresponds to the specific ORFs on the
array and the ORFs are arranged in the genome order of the reference
strain 8004 from XC0001 at the top to XC4332 at the bottom. From the
aCGH result, it is observed that the ATCC33913 is missing two
prominent DNA fragments, one from strain 8004 ORF XC2030 to
XC2074 and the other from XC2399 to XC2444, which is consistent with
on 16S-23S rDNA ITSs showed a similar grouping pattern in
hierarchical clustering (Figure 2 and Additional data file 2).
However, no significant relationship was observed between
phylogenetic group and pathogenicity, or pathogenicity and
No significant correlations were observed between the gross
genome composition of Xcc strains and their pathogenicity,
or the genome composition of the strains and their
geographical origins. However, strains CN14, CN15, and CN16, which
were isolated from different host plants around Guilin city,
are significantly conserved in genome composition and
exhibit similar pathogenicity (Tables 1 and 2; Additional data
file 4). This suggests that the three strains may share a most
recent common ancestor that is different from that (those) of
the other Chinese strains.
The variable genomic regions and their divergence in
The locations of the variable genes in the different strains
identified by the aCGH analysis were mapped onto the
chromosome of strain 8004. The results revealed that there are 27
such chromosomal regions, each of which consists of more
than three contiguous CDSs in the 8004 genome (Figure 2).
These regions were named XVRs for Xanthomonas variable
genomic regions and numbered from 1 to 27 in accordance
with the genome coordinates of strain 8004 (Table 6). The
boundaries of the XVRs were determined at the CDS level, to
fit in with the resolution of the array hybridization analysis in
this study. The 27 XVRs contain 402 CDSs and account for
48.4% of the AHD genes, representing 9.41% of the total
CDSs of Xcc strain 8004.
The size of the XVRs ranges from 1,778 bp (XVR24 with only
three CDSs) to 98,358 bp (XVR13 with 81 CDSs) (Table 6).
There are 15 XVRs larger than 10 kb and 4 larger than 50 kb.
Within the XVRs, there are 27 genes encoding proteins for
pathogenicity and adaptation, 9 for regulatory functions, 25
for cell structure and cell processes, 19 for intermediary
metabolisms, 95 for mobile elements, 21 for DNA metabolism
related to mobile elements, and 219 encoding hypothetical or
function-unknown proteins (Table 6 and 7).
The distribution patterns of XVRs show significant diversity
among the Xcc strains tested (Table 8). Five XVRs (XVR02,
XVR17, XVR18, XVR20 and XVR27) are AHD from all the
Chinese strains tested (Table 8). XVR17 and XVR18 are also
absent from the British strain ATCC33913 as pointed out by
Qian et al. . Most of the genes in these five XVRs encode
hypothetical proteins for which there are no significantly
similar sequences in GenBank.
XVR04 is a typical integron, which contains a gene for a DNA
integrase (intI) catalyzing the site-specific recombination of
gene cassettes at the integron-associated recombination site
(attI), and a cassette array of 14 genes with unknown function
[21,46]. Integrons are best known for assembling antibiotic
resistance genes in clinical bacteria. They capture genes by
integrase-mediated site-specific recombination of mobile
gene cassettes. It has been postulated that the ancestral
xanthomonad possessed an integron at ilvD, an acid dehydratase
gene flanking the intI site-specific recombinase . The
The number of conserved and absent/highly divergent CDSs in Xcc strains
CDSs on chip
*Altogether, 730 CDSs were AHD among the Chinese strains, of which 58 were commonly AHD in all the Chinese strains. Fifty-one CDSs were
found to be given invalid results.
microarray results showed that all of the Chinese strains
tested possess the ilvD gene, although whether its
organization is conserved in these strains is unknown. However,
significant diversity found in the integron cassette array among
these Chinese strains suggests that the integron might also
generate diversity within the pathovar, in addition to between
XVR14 contains 21 CDSs with two copies of the phi Lf-like
Xanthomonas prophage, which harbors the putative dif site
of replication termination of the Xcc strains 8004  and
Xc17 . In strain ATCC33913, the two copies of Lf-like
prophage possess the typical genetic organization of
filamentous phages, that is, a symmetrical head-to-head
constellation, with genes functioning in DNA replication, coat
synthesis, morphogenesis and phage export . In strain
8004, only one copy of the Lf-like prophage is intact and the
other lacks two genes (gII and gV) [20,21]. This phi Lf-like
prophage is missing from or highly divergent in most of the
Chinese strains tested and most other xanthomonads
sequenced, but present in Xcv 85-10  (Table 9 and Figure
3). It is worth mentioning that the P2-like prophage ,
which occurs in strain ATCC33913 but is missing from strain
8004, is found to be AHD from all of the Chinese strains
tested by hybridization analysis using a probe from
There are two clusters of the type I restriction-modification
system in strain 8004, of which one is present in strain
ATCC33193 and the other is unique to strain 8004 [20,21].
XVR22 is one of these clusters. In contrast to ATCC33913,
which lacks this locus, most of the Chinese strains possess it.
Restriction and modification systems are responsible for
cellular protection and maintenance of genetic materials against
invasion of exogenous DNA. There is evidence that they have
undergone extensive horizontal transfer between genomes, as
inferred from their sequence homology, codon usage bias and
GC content difference. In addition to often being linked with
mobile genetic elements, such as plasmids, viruses,
transposons and integrons, restriction-modification system genes
themselves behave as mobile elements and cause genome
XVR23 consists of 14 ORFs that contains several genes for
lipopolysaccharide (LPS) O-antigen synthesis, including
wxcC, wxcM, wxcN, gmd and rmd , which is discussed
below. Some predicted functions of other XVRs are shown in
Table 7 based on the annotation of their component CDSs.
Horizontal gene acquisition and gene loss
The detection of DNA segments in which integrase genes are
associated with tRNA or tmRNA genes [51-53], or regions of
anomalous GC content with mobile elements , facilitates
C01 Amino acid biosynthesis
C02 Biosynthesis of cofactors, prosthetic groups, carriers
C03 Cell envelope and cell structure
C04 Cellular processes
C05 Central intermediary metabolism
C06 Energy and carbon metabolism
C07 Fatty acid and phospholipid metabolism
C08 Nucleotide metabolism
C09 Regulatory functions
C10 Replication and DNA metabolism
C14 Mobile genetic elements
C15 Putative pathogenicity factors
C15.01 Type I secretion system
C15.02 Type II secretion system
C15.03 Type III secretion system
C15.04 Type IV secretion system
C15.05 Type V secretion system
C15.06 Sec and TAT system
C15.07 Type III-effectors and candidates
C15.08 Host cell wall degrading enzymes
C15.12 Toxin and adhesin
C15.13 Quorum sensing
C15.14 Other pathogenicity factors
C16 Stress adaptation
C17 Undefined category
C18 Hypothetical proteins
Distribution of strain 8004's CDSs and the AHD CDSs by functional categories
the identification of horizontally acquired sequences in
genomes. Horizontally acquired sequences are also
detectable by comparing their dinucleotide composition (genome
signature) dissimilarity (* value) with that of the host
genome. The higher * values of XVRs can be indicative for
horizontal acquisition . The data presented in Tables 6
and 7 show that XVR09, XVR13, XVR18 and XVR19 are
integrated adjacent to or within tRNA genes with an integrase or
insertion sequence (IS) flanking the ends. XVR04, an
integron , and XVR14, a phi Lf-like prophage [20,21], are also
actively transferred DNA sequences. Obviously, the five
XVRs, XVR02, XVR17, XVR18, XVR20 and XVR27, which are
ubiquitously AHD from all the Chinese strains tested, could
be the most recently acquired DNA in strain 8004. It is
possible that the donors of these five XVRs are probably absent in
mainland China. In contrast, we consider that the XVRs
present in the other sequenced xanthomonad strains may be
a result of acquisition events during the early stage of
Xanthomonas evolution and lost from certain Xcc strains at a
later stage, probably due to DNA deletion events.
The identification of Xcc DNA loss events can be carried out
by analysis of the sequenced xanthomonads for the presence
of collinear blocks that encompass the targeted DNA
segments. Whole genome comparisons among Xcc 8004 ,
Xcc ATCC33913 , X. axonopodis pv. citri 306 , X.
campestris pv. vesicatoria 85-10 , X. oryzae pv. oryzae
KACC10331  and X. oryzae pv. oryzae MAFF311018 ,
CG% f800o4 nogeem 8004 33913 07CN 03CN 01CN 11CN 02CN 12CN 08CN 04CN 17CN 06CN 18CN 14CN 15CN 16CN 05CN 09CN 10CN 20CN
SoFcnighaeuCmreGatH2ic arneaplryesseesntation of the genome composition of Xcc strains based
Schematic representation of the genome composition of Xcc strains based
on aCGH analyses. The left-most line indicates the physical map scaled in
megabases from the first base, the start of the putative replication origin.
The curve indicates the GC content in the genome of strain 8004. The
image of the hierarchical clustering was based on the aCGH results of 20
Xcc strains. The number of Xcc strains on the top shows that each column
indicates each strain. Each tiny line indicates a specific CDS on the array,
and the CDSs are arranged in the order of the genome of strain 8004.
Each green line indicates an AHD CDS in the corresponding test strain.
The serial numbers on the right indicate the variable genomic regions of
allowed identification of a number of XVRs (XVR03, XVR05,
XVR08, XVR10, XVR11 and XVR22) as DNA segments
inherited from the common ancestral xanthomonad (Figure 3). In
each case, large DNA segments containing each of these XVRs
have a high degree of synteny in other xanthomonads (Figure
3 and Table 9).
Analysis of the structure of XVR13 and its distribution pattern
in Xcc strains revealed that this region might undergo a series
of multiple insertion and deletion events during the Xcc
evolution (Figure 4). This region is near the terminus of
chromosome replication, which is susceptible to gene acquisition
and/or gene loss . XVR13 is the largest genomic island
identified in Xcc 8004, which spans nucleotide coordinates
from 2,414,668 to 2,513,025 and contains 81 CDSs. To its left
flank are three tRNA genes and an integrase gene. Genome
comparison showed that the central part of XVR13, named
XVR13.1, is totally absent in strain ATCC33913. XVR13.1 is
58,007 bp in length. The aCGH results reveal that three
Chinese strains (CN01, CN03 and CN11) contain the XVR13
locus, which is almost identical to that of Xcc 8004, and four
Chinese strains (CN07, CN09, CN10 and CN20) contain an
incomplete XVR13 locus without XVR13.1 that is almost
identical to that in strain ATCC33913, and the rest of the Chinese
strains probably have no XVR13 (Table 8 and Figure 4). To
elucidate the dynamic relationship between XVR13 and
XVR13.1, re-annotation was done for XVR13.1 and 63 CDSs
were identified (Figure 4 and Additional data file 5). A
truncated yeeA-like gene was found across the right border of
XVR13.1 (Figure 4). Intriguingly, yeeB- and yeeC-like genes
occur in both Xcc strains 8004 and ATCC33913 (Figure 4).
This suggests that XVR13.1, or at least part of it, has been lost
from the British strain ATCC33913 and most of the tested
Chinese strains during their evolution.
XVR23, part of the wxc cluster, contains several genes for
Oantigen synthesis of LPS . The aCGH results revealed that
this region is highly divergent, with a mosaic structure among
the Chinese strains tested. Sequence comparisons showed
that wxc cluster of Xcc 8004 is significantly divergent from
that of Xcc B100 , although it is almost identical to that of
Xcc ATCC33913 [20,21]. The wxc cluster of strain 8004 is
truncated by IS elements and some of the wxc genes have low
similarity to the corresponding genes of strain B100.
Significant differences in wxc clusters among other
xanthomonad strains have also been reported [48,56,58]. The
Xcc wxc cluster not only has a significantly lower GC content
(56.82%) than the average genome level (64.95%), but also
has a very high * value of 81.182. These suggest that Xcc
might have acquired the wxc cluster by horizontal DNA
The distribution of pathogenicity-related genes among
Bioinformatic analysis revealed that strain 8004 contains 197
CDSs that show homology to the confirmed or annotated
putative pathogenicity genes of plant or animal pathogenic
bacteria, in addition to 108 genes that have been proven to be
involved in Xcc pathogenicity (Additional data file 6). Of
these 305 proven or presumed pathogenicity genes, 304 were
spotted on the microarray slides of strain 8004 in this study.
The other CDS (XC3591) encoding pectate lyase was not
spotted as it has a redundant DNA sequence in the genome of
strain 8004. The aCGH analysis revealed that 258 of the
pathogenicity genes (84.8% of the pathogenicity genes spotted)
are present in all of the Xcc strains tested and 46 (15.1%) are
AHD in at least one of the strains (Table 5 and Additional data
file 6). The results show that the pathogenicity genes involved
The variable genomic regions in strain 8004
These variable genomic regions (XVRs) are totally absent from the genome of Chinese strains. XVR13.1 denotes that the fragment is a part of
in the type I, II and III secretion systems (T1SS, T2SS and
T3SS), host cell wall degradation, extracellular
polysaccharide production, and the quorum sensing system are highly
conserved in almost all of the Xcc strains tested (Table 5 and
Additional data file 6). In addition, genes encoding proteins
of the gluconeogenic pathway , Mip-like protein , the
catabolite repressor-like protein Clp , and zinc uptake
regulator protein Zur , which have been demonstrated to
play important roles in Xcc virulence, are also highly
conserved. However, genes relating to T4SS, T3SS-effectors and
candidates, LPS synthesis, toxin as well as adhesin are highly
diversified (Table 5 and Additional data file 6).
LPS is an indispensable component of the cell surface of
Gram-negative bacteria and has been demonstrated to play
important roles in pathogenicity of several phytopathogenic
bacteria, including Xcc [62-64]. More than 20 genes for LPS
synthesis have been characterized in Xcc. These include
xanAB , rmlABCD , rfaXY , lpsIJ  and the
wxc cluster consisting of 15 genes . The aCGH results
suggest that lpsIJ, rfaXY, rmlABCD and xanAB are highly
conserved while wxc genes are divergent in the Xcc strains
tested. The wxc genes are involved in the biosynthesis of the
LPS O-antigen, which is the most variable portion of LPS
[19,68]. The diversity of the wxc cluster indicates that the
LPSs produced by Xcc different strains may be varied.
T4SSs have been validated as having important roles in the
pathogenesis of several animal and plant bacterial pathogens
[36-38,40]. The T4SS of Agrobacterium tumefaciens is
essential for virulence and is assembled from the proteins
encoded by the virB cluster and virD4. Many T4SSs are
highly similar to the A. tumefaciens VirB/D4 T4SS .
Burkholderia cenocepacia strain K56-2 can produce the plant
tissue watersoaking phenotype (a plant disease-associated trait)
and possesses two T4SSs similar to the VirB/D4 system .
The characteristics of the variable genomic regions in strain 8004
Regulatory protein cII, putative secreted proteins
Deoxycytidylate deaminase and Rhs protein, genes related T4 phage
Integron, xanthomonadin biosynthesis
Type I site-specific deoxyribonuclease
ThiJ/PfpI family protein, oxidoreductase
Transcriptional regulator BlaI family
Regulatory protein BphR
Fimbrial assembly protein
Type IV pilin
VirB cluster for T4SS
Avirulence proteins, pathogenicity related proteins
Adaptation, virulence related protein
Histidine kinase/response regulator hybrid protein, single-domain response
Nucleotide sugar transaminase
IS elements Arsenite efflux, iron uptake
Integrase + tRNA-Arg IS elements Plasmid mobilization protein, hemolysin activation protein
Integrase + tRNA-Ser IS elements Avirulence protein, phage related protein
IS elements Integrase Phage related protein, helicase
Type I site-specific restriction-modification system, virulence protein
Sugar translocase, O-antigen
Avirulence protein, regulators
Rich in mobile elements
Occurrence of XVRs*
Mutational studies in B. cenocepacia strain K56-2 revealed
that the plasmid-encoded T4SS is involved in eliciting the
plant tissue watersoaking phenotype and responsible for the
secretion of a plant cytotoxic protein(s), while the
chromosome-encoded T4SS is not . Genome annotation revealed
that the Xcc strain 8004 has an A. tumefaciens VirB/D4-like
T4SS . Although genomic sequence comparison showed
that the Xcc strain ATCC33913 possesses an almost identical
virB cluster to that of strain 8004, the aCGH analyses
displayed that the virB cluster of most Chinese strains tested is
AHD. Since all these strains were fully virulent and their
aCGH intensity ratios were extremely low (as low as
0.10.025; Additional data file 4), a query on the role of the T4SS
in Xcc pathogenicity was raised. To answer this question, we
constructed a T4SS mutant derived from strain 8004 (Figure
5). A mutant with deletions of the virB cluster as well as virD4
was confirmed by PCR and designated 8004T4 (Figure 5
and Additional file 7). The virulence of the mutant was tested
on host plants cabbage (B. oleracea var. capitata) cv.
Jingfeng-1, Chinese cabbage (B. rapa subsp. pekinensis) cv.
Zhongbai-83, Chinese kale (B. oleracea var. alboglabra) cv.
Xianggangbaihua, pakchoi cabbage (B. rapa subsp.
chinensis) cv. Jinchengteai, and Radish (R. sativus var. radicula) cv.
Manshenghong by the leaf-clipping inoculation and spray
methods. The results showed that the virulence of the mutant
was as severe as on the wild type strain 8004 on all the tested
plants inoculated by leaf-clipping (Figure 5) or spray (data
not shown). This suggests that the T4SS is not involved in the
virulence of Xcc.
The genetic determinants for host specificity of Xcc
Genes involved in the host specificity of Xcc are of central
interest in this study. All of the Xcc strains used in this work
are able to cause disease in their host plants but show
The distribution of variable genomic regions in Xcc strains
+, the XVR is present; -, AHD; (+), some CDSs of the XVR might be present and are ordered in the allele in the given genome; (-), a few CDSs of the
XVR are scattered in the allele in the given genome.
icity for a host range. Apart from four strains (CN01, CN04,
CN05 and CN17) that could infect all of the host plants tested,
the other 16 strains were avirulent on certain host plant(s)
(Table 2). The host specificity of pathogens is determined by
gene-for-gene interactions  involving avirulence (avr)
genes of the pathogen and cognate resistance (R) genes of the
host. Disease resistance occurs in a host-pathogen interaction
in which an R gene in the host is matched by a cognate avr
gene in the challenging pathogen. A pathogen-host
interaction without such a cognate avr-R combination will
lead to disease.
To elucidate the genetic determinants for host specificity of
Xcc, the correlation between the virulence scale on host
plants and the gene distribution pattern of the 20 Xcc strains
was analyzed. The correlation between HR induction on
nonhost plants and gene distribution patterns of the strains was
also determined. Twelve operations were performed and the
correlation coefficient (CC) values of these are given in
Additional data files 8 and 9. Seven of the eleven host plants are
susceptible to all of the 20 Xcc strains tested (Table 2),
indicating that they have no CC values. Correlation analyses
for the other four host plants and one non-host plant
discovered four candidate genes responsible for the
virulence-deficiency (negative CC value) of Xcc strains on a particular host
plant(s) and one candidate for HR induction (positive CC
value) on the non-host plant pepper ECW10R. These genes
are candidates of the three postulated avr genes avrRc1,
avrRc3 and avrRp1 (Table 10). The candidates XC2004 and
XC2084 are correlative to avrRc3 and have the same CC
value. XC2084 encodes a transposase , suggesting that its
postulated avrRc3 is much smaller than that of XC2004.
Therefore, XC2084 was removed from the candidate list. The
candidate genes XC2602, XC2004 as well as XC2081 have
been annotated as encoding Avr-homologous proteins .
To identify avr genes from the candidates, we further
investigated their biological functions by mutagenesis. The
candidate avr genes of Xcc 8004 were disrupted by using the
plasmid pK18mob , a conjugative suicide plasmid in Xcc
(see details in Materials and methods). The obtained
nonpolar mutants of XC2602, XC2004 and XC2081, named
The distribution of variable genomic regions in other sequenced Xanthomonas spp.
Figure 3 (see legend on previous page)
X X X
ccC locus dominates the avirulence of Xcc on mustard plants.
A X X tR tR tR 0 c 0 5 8 5 0 7 7 9 1 1 1 1 1 1 2 2
C C 3 C 5 9 3 4 5 7 8 9 0 1
0 c 0 9 7 95 02 74 74 74 02 02 02 6B 02 02 20 0 0
S 2 rX 2 5 4 2 2
t C v C 1 1 1 C 1 1 1 C C C ir C C C C C
in X a X IS IS IS X IS IS IS X X X v X X X X X
C C N N N
X X tR tR tR
a IS IS IS IS IS IS
X X X X ye ye ye ye X a a I T X D X X X
B C s1B rs1B c1d 410
core genome integrase gene IS element divergent gene
yee gene no array more genes
TFihgeuprrees4umed allelic loci of XVR13 in the Chinese Xcc strains suggested by aCGH
CN15, CN16 and CN18; (h) strains CN14 and CN17. IS, insertion sequence.
The genetic structure of virB/D4 locus in Xcc 8004
The deleted region (coordinates 1957097~1965913)
virB/D4 locus in deleted mutant
To verify the avirulence function of Xcc avrXccE1 (XC2602),
the cosmid pLAFR6 carrying a PCR-generated 1,605 bp
fragment encompassing the region 514 bp upstream of the start
codon to 29 bp downstream of the stop codon of XC2602 was
introduced by triparental mating into the Chinese strains
CN01, CN05, CN10 and CN11, which showed virulence on
Chinese cabbage cv. Zhongbai-83 (Table 2). The obtained
transconjugants for all the four strains lost virulence on
Identification of the genetic determinants for host specificity of Xcc 8004 on certain plants
Candidate avr genes in Xcc 8004
Postulated avr gene
Avirulence protein, avrXccC
Avirulence protein, avrXccE1
Avirulence protein, avrBs1 gene
Chinese cabbage cv. Zhongbai-83 (Figure 7). These results
demonstrate that avrXccE1 (XC2602) of Xcc is endowed with
an avr function determining host specificity.
In this work, we constructed a whole-genome microarray
based on the determined genome sequence of Xcc strain
8004 isolated in the UK and used it to explore by aCGH
analyses the genome contents and gene diversity among 18 Xcc
strains isolated from different host plants and various
geographical regions over a wide range of latitudes across China.
Several attractive divergent genetic determinants related to
pathogenicity uncovered by aCGH analyses were further
functionally characterized, enabling the discovery of avr
genes affecting Xcc host specificity and the T4SS that are not
involved in symptom production by Xcc.
Our aCGH analyses revealed that 3,405 (81.3%) of the 4,186
genes of the Xcc strain 8004 spotted on the array were
conserved in all the 18 Chinese Xcc strains tested. These
conserved genes represent a rough genetic core of Xcc. This
percentage is much higher than the 53% observed in 17
strains of the phytopathogenic bacterium Ralstonia
solanacearum . The Xcc core content contains not only
the genes for essential metabolism, but also the genes
encoding the main pathogenicity factors (see below) and
proteins involved in xanthomonadin biosynthesis. The aCGH
analyses also revealed that the Xcc strains possess a flexible
gene pool of 730 CDSs, accounting for 17.6% of all the valid
hybridized 4,135 CDSs in the aCGH analyses of all 18 Chinese
strains. These genes are AHD from the Chinese strains
compared with the reference strain 8004. The number of AHD
CDSs of individual strains ranges from 137 to 475, which is
more than the 108 strain-specific genes of Xcc 8004
compared to strain ATCC33913 and the 62 strain-specific genes of
ATCC33913 compared to Xcc 8004, revealed by comparison
of the two strains' whole genome sequences [20,21]. Among
the 730 flexible genes, 58 are AHD from all the Chinese
strains. Of these, 57 are situated in eight XVRs while one is
alone; 42 located mainly in XVR13.1, XVR17 and XVR18 are
also absent from the British strain ATCC33913 . Whether
the remaining 16 ADH CDSs in XVR02, XVR14, XVR20,
XVR23 and XVR27, which are conserved in the British strains
8004 and ATCC33913, constitute the major genetic
differences between British Xcc strains and Chinese Xcc strains
needs further studies on more strains. Most of the 27 XVRs
possess DNA sequences associated with integrase genes or
mobile elements and with lower GC content and higher *
value compared to Xcc regular genomic characteristics,
implying that these DNA sequences may have been acquired
through horizontal gene transfer [53,72].
Since all the strains used in this study are fully virulent in
certain host plants, the genetic core revealed by aCGH
characterization of these strains should cover the pathogen's
symptom production and the basic pathogenicity
determinants of the pathogen; hence the flexible genes might not be
essential for virulence of the pathogen. The leaf-clipping
inoculation method used for the pathogenicity tests in this study
directly delivers bacterial cells into the vascular system of the
host plant. Some of the genes involved in the early stages of
the interaction between the pathogen and the host might be
concealed in the flexible gene pool.
Eight avr genes are annotated in the genome of both Xcc
strains 8004 and ATCC33913 based on their sequence
homology to avr genes identified in other pathogens [20,21]. It
has been shown that mutagenesis of all the eight avr genes in
Xcc strain 528T (ATCC33913) has no detected effect on
virulence and only one of the avr genes affects race specificity
. However, it has been proposed that Xcc is composed of
6 races, based on the interactions of 144 isolates with 6
different host varieties in the 4 Brassica species B. carinata, B.
juncea, B. oleracea and B. rapa . The 20 strains used in
this study could be grouped into three races based on their
FVirguulernec6e and HR of Xcc strains
Virulence and HR of Xcc strains. (a) Xcc 8004 and the complementary
strain CNK2004 are avirulent, but the mutant NK2004 is virulent on the
host plant, mustard (B. juncea var. megarrhiza Tsen et Lee) cv. Guangtou.
The photographs were taken ten days after inoculation. (b) Xcc 8004 and
the complementary strain CNK2602 are avirulent, but the mutant
NK2602 is virulent on the host plant, Chinese cabbage (B. rapa subsp.
pekinensis) cv. Zhongbai-83. The photographs were taken ten days after
inoculation. (c) Xcc 8004 and the complementary strain CNK2081 could
induce HR, but the mutant NK2602 and the negative control 8004hrpG
could not induce HR on the non-host plant, pepper (Capsicum annuum) cv.
ECW10R. The photographs were taken 24 h after inoculation.
disease reactions on nine host varieties (or subspecies) in
three Brassica species, B. junccea, B. oleracea and B. rapa, as
well as the Raphanus species R. sativus. In addition, eight
strains, including 8004 and ATCC33913, could induce HR on
the non-host plant pepper ECW10R carrying the R gene Bs1
, indicating that these strains harbor a cognate avr gene
avrBs1. To identify the avr genes in Xcc strain 8004, we
employed a correlation analysis between the strain-plant
reaction and the gene distribution pattern of strains to screen
avr candidates and then ascertained the avirulence function
of the candidates by genetic experiments. This strategy
allowed us to identify the avr genes avrXccC, avrXccE1 and
avrBs1. The avrXccC gene of strain 8004, conferring
avirulence on mustard cultivar Guangtou, is identical with the
avrXccFM of strain 528T (ATCC33913), conferring avirulence
on Florida mustard . This study verified that avrXccE1
affects host specificity by conferring avirulence on Chinese
cabbage cv. Zhongbai-83. The avrXccE1 of strain 8004 is
identical to the XCC1629 of strain 528T. These two strains
showed incompatible reactions on Chinese cabbage cv.
Zhongbai-83 (Table 2). Castaeda and associates  did not
observe such an avirulence function for XCC1629 of strain
528T on Early Jersey Wakefield cabbage, suggesting that the
R gene responsive to avrXccE1 (XCC1629) exists in Chinese
cabbage cv. Zhongbai-83 but not in Early Jersey Wakefield
cabbage. The avrBs1 of strain 8004 was validated to be
responsible for eliciting HR on the non-host plant pepper
ECW10R. The sequences of avrBs1 of strain 8004 and
XCC2100 of strain 528T (ATCC33913) are exactly the same
[20,21]. Both strains could induce HR on the pepper ECW10R
(Table 2) . However, Castaeda and associates did not
detect HR variation between the XCC2100 mutants and the
wild-type 528T . It is possible that the function of avrBs1
is redundantly encoded in 528T and that the expression and
regulation of avrBs1 and XCC2100 in 8004 and 528T
(ATCC33913) is different. The postulated avr gene avrRc2
exists in the strains ATCC33913, CN14, CN15 and CN16 but
not in the aCGH reference strain 8004. Work to identify
avrRc2 from the ATCC33913-strain specific CDSs (compared
to strain 8004) are underway.
Avirulence genes have been generally identified by molecular
genetic methods where clones from a genomic library of an
avirulent strain are mobilized into a virulent strain and the
resulting transformants or transconjugants are tested for an
alteration in the outcome of the pathogen-host interaction
[74-77]. Genomic mining has also provided a powerful tool to
uncover avr genes by homology searches and bioinformatic
approaches [78-80]. Comparatively, a major advantage of the
aCGH approach in identifying host specificity genes is the
high-throughput and efficiency at identifying genome
diversity at the gene level. This allows parallel identification of
candidate genes for a number of avirulence determinants
through the correlation analysis between the phenotype
(avirulence/virulence) and the gene distribution pattern in a
bacterial strain population. It could be expected that analysis of
an increased number of strains in parallel with virulence
assays on an increased number of host plants will enhance a
full-scale identification of host specificity genes from a
pathogen. The main limitations of the current aCGH approach are
that it satisfies only the analysis of genes present in the
reference strain and that it is incapable of identifying single
nucleotide polymorphisms that may also contribute to gene
Our aCGH results revealed that 258 (84.8%) of the 304
proven or presumed pathogenicity genes are conserved in all
the Xcc strains tested and 46 (15.1%) are AHD. A large portion
of these AHD genes are the wxc genes and the genes encoding
T4SS as well as T3SS-effectors. The wxc gene cluster is
involved in the synthesis of the LPS O-antigen. In Xcc, LPS
has been demonstrated to play important roles in
pathogenicity  and disruption of the wxc genes resulted in significant
reduction of virulence . As all the Xcc strains used in this
study are fully virulent at least on some of the host plants
tested, these strains may not be defective in LPS production.
The diversity of the wxc genes suggests that the LPSs
synthesized by Xcc different strains may have different structures.
Among plant bacterial pathogens, the role of T4SS in
pathogenicity has not been experimentally verified except in A.
tumefaciens , although T4SSs have been annotated as
putative pathogenicity-related machines in the genomes of
many pathogens, including the Xcc strains ATCC33913 and
8004 [20,21]. The high divergence of T4SS among the
virulent Xcc strains revealed by the aCGH analyses prompted
us to validate the role of T4SS in Xcc pathogenicity by genetic
experiments. The T4SS in strain 8004 is encoded mainly by
virD4 and the virB cluster, which consists of nine ORFs .
Deletion of virD4 and the virB cluster of strain 8004 did not
affect the virulence of the pathogen on all the host plants
tested, indicating that the T4SS is not engaged in the
pathogenicity of Xcc. What is the function of T4SS in Xcc? Is it
involved in bacterial conjugation and/or effector
translocation? This will be the subject of future studies. Genomic data
show that an entire T4SS encoded by virD4 and the virB
cluster also exists in the phytopathogenic bacteria Erwinia
carotovora , Pseudomonas syringae pv. phaseolicola , R.
solanacearum , X. axonopodis pv. citri , X.
campestris pv. vesicatoria  and X. fastidiosa . To investigate
experimentally the role of the T4SS in the pathogenicity of
these pathogens will no doubt facilitate the understanding of
the T4SS functions in plant bacterial pathogenesis.
The results of our aCGH analyses reveal that about 80% of
CDSs (3,405 CDSs) are conserved among 20 different
virulent strains of Xcc. These conserved CDSs may stand for the
core genome of Xcc, although the variable genes will increase
in quantity with more strains to be analyzed. The core genome
includes not only house-keeping genes but also a large
amount (258) of proven or presumed pathogenicity-related
genes. This work has also demonstrated that the T4SS, which
has been validated to play important roles in the pathogenesis
of a number of animal and plant bacterial pathogens and
predicted to be a pathogenicity-related machine in many
bacterial genomic annotations, is not involved in the pathogenicity
of Xcc. Compared to the reference strain 8004, the number of
flexible genes of individual Chinese Xcc strains ranges from
137 to 475. The wxc gene cluster, which is involved in LPS
Oantigen synthesis and the pathogenicity of Xcc, is highly
divergent among different Xcc strains. It is possible that the
LPSs synthesized by different Xcc strains have various
structures. We show an efficient strategy to identify avr genes
determining pathogens' host specificities. Three avr genes
from the Xcc strain 8004 were identified by the application of
this strategy in this study. More avr genes in the Xcc strain
8004, if present, could be discovered by this approach with
more different host plants.
Materials and methods
Bacterial strains, culture conditions and molecular
Xcc isolates used in this study were collected from various
geographical locations over a wide range of latitudes across
mainland China (Tables 1 and 2). The bacteria were isolated
from the infected leaves of cruciferous plants with typical
symptoms of black rot disease. Recovered colonies were
picked and re-streaked onto NYG  agar plates to verify the
bacterial identity. Each isolate was inoculated onto radish (R.
sativus var. radicula) cv. Manshenhong by the leaf clipping
method  to evaluate its pathogenicity. The 16S-23S rDNA
ITS was amplified as described by Gurtler and Stanisich 
using primer R1 and primer R2 (Additional data file 1).
Molecular manipulations, genomic DNA preparations,
restriction endonuclease digestions and PCR amplifications
were performed as described by Sambrook et al. .
Enzymes were supplied by Promega (Shanghai, China) and
used in accordance with the manufacturer's instructions.
The virulence of Xcc strains was evaluated on 11 host plants:
cabbage (B. oleracea var. capitata) cv. Jingfeng-1, Chinese
cabbage (B. rapa subsp. pekinensis) cv. Zhongbai-4, Chinese
cabbage (B. rapa subsp. pekinensis) cv. Zhongbai-83,
Chinese kale (B. oleracea var. alboglabra) cv. Xianggangbaihua,
kohlrabi (B. oleracea var. gongylodes) cv. Chunqiu, mustard
(B. juncea var. megarrhiza Tsen et Lee) cv. Guangtou,
pakchoi cabbage (B. rapa subsp. chinensis) cv. Jinchengteai,
pakchoi cabbage (B. rapa subsp. chinensis) cv. Naibaicai,
radish (R. sativus var. sativus) cv. Cherry Belle, radish (R.
sativus var. longipinnatus) cv. Huaye, and radish (R. sativus
var. radicula) cv. Manshenhong (Table 2). All of these
cultivars are available from the Institute of Vegetables and
Flowers, Chinese Academy of Agricultural Sciences, Beijing
100081. Each Xcc strain was tested on all the 11 cultivars. The
bacteria grown overnight in NYG medium  were washed
and resuspended in water to a cell density OD of 0.01 at 600
nm. The last completely expanded leaf of the five-week old
seedlings was inoculated by cutting with scissors dipped in
bacterial suspensions  or by spraying the bacterial
suspensions with a sprayer. Twenty leaves were inoculated for
each strain-plant combination. The inoculated plants were
kept in a culture room at a temperature of 28C and a relative
humidity of 80% under 16 h light day, after 24 h moisture
preservation in a plastic chamber at a temperature of 28C
and a relative humidity of about 100%. First symptoms
appeared five days post-inoculation, and the lesion lengths of
20 leaves were measured 10 days post-inoculation for each
strain-plant combination. The virulence of each Xcc strain on
each host plant was rated according to the disease symptoms:
non-pathogenic, leaves with no visible effect, or with localized
necrosis (HR) or with few small lesions (less than 3 mm) near
cuts; weakly virulent, leaves with chlorosis extending from
cuttings; and fully virulent, blackened leaf veins, death, and
drying of tissue with V-shape lesions. This rating method was
modified from Ignatov et al. .
For HR tests, Xcc strains were cultured as for the virulence
assay, adjusted to a density of 108 colony forming units per ml
with distillated water and introduced, by the infiltration
method with a needleless syringe , into the intercellular
spaces of the leaves of non-host plant pepper (Capsicum
annuum cv. Early Cal Wonder) ECW10R (from Laboratoire
de Biologie Moleculaire des relations Plantes
Microorganismes INRA-CNRS, Castanet Tolosan, France).
After inoculation, the plants were kept at 28C under
continuous illumination of 6,000 lux light intensity. The hrpG
mutant, a Xcc deletion mutant of hrpG , was used as a
Construction of the whole-genome microarray of Xcc
A high-density PCR-based DNA array was designed by using
the genome sequence data of Xcc strain 8004 (GenBank
accession number CP000050). The genome has 5,148,708 bp
and encodes 4,273 predicted CDSs . An in-house
highthroughput computer algorithm based on the Linux
operating system and Python programming language was employed
to design PCR primers for all CDSs. The fundamental rules of
our computer algorithm include that all the primer annealing
temperatures range from 57.5-68.7C, and the PCR product
sizes fall within 200-1,000 bp, with an optimum of 500 bp.
The PCR amplicons should have a minimum sequence
similarity with cut-off e-value <1 e-3 and sequence identity <70%
when using the BLAST program. There are 87 genes which
were designed not to be spotted on the array because of their
high sequence similarity to other genes in the genome. The
PCR amplifications were performed in a 100 l reaction
volume and PCR success was confirmed by agarose gel
electrophoresis. The confirmed PCR products were precipitated with
isopropanol and redissolved in DNA Spotting Solution
(CapitalBio Corp., Beijing, China). For ORFs that were too small or
those genes for which PCR amplification failed, 70-mer sense
oligonucleotides were designed; 143 such oligonucleotide
probes were synthesized. PCR products and 70-mer
oligonucleotides (20 M) were printed on amino silaned glass slides
(CapitalBio Corp.) using a SmartArray microarrayer
(CapitalBio Corp.). Each CDS was printed in triplicate to
facilitate subsequent data analysis. After printing, the slides were
baked at 80C for 1 h and stored dry at room temperature till
use. The Xcc 8004 microarray slides are available to the
public from CapitalBio Corp. .
Prior to hybridization, the slides were rehydrated over 65C
water for 10 s, and UV cross-linked at 250 mJ/cm2. The
unimmobilized DNAs were washed off with 0.5% SDS for 15
minutes at room temperature and SDS was removed by
dipping the slides in anhydrous ethanol for 30 s. The slides were
spin-dried at 1,000 rpm for 2 minutes.
DNA labeling for aCGH analysis
Genomic DNA was fragmented by Dpn II endonuclease
digestion, and then purified with the PCR Clean-up NucleoSpin
Extract II kits (Macherey-Nagel, Dren, Germany). For each
labeling reaction, 2 g of digested DNA and 4 g of random
nonamer were heated to 95C for 3 minutes and snap cooled
on ice, then 10 buffer, dNTP and Cy5-dCTP or Cy3-dCTP
(GE HealthCare Bio-Sciences AB, Bjrkgatan, Uppsala,
Sweden) were added at final concentrations of 120 M each dATP,
dGTP, dTTP, 60 M dCTP and 40 M Cy-dye. Klenow enzyme
(1 l; Takara, Dalian, China) was added and the reaction was
performed at 37C for 1 h. The labeled DNA was purified with
a PCR Clean-up NucleoSpin Extract II kit, resuspended in
elution buffer and checked for its optical density.
Microarray hybridization, scanning and data analysis
For aCGH, the final products hybridized with microarrays
were fluorescence-labeled DNA, so an identical hybridization
strategy was employed. Labeled control and test samples
were quantitatively adjusted based on the efficiency of Cy-dye
incorporation and mixed into 80 l hybridization solution
(3 SSC, 0.2% SDS, 50% formamide). DNA in hybridization
solution was denatured at 95C for 3 minutes prior to loading
on the microarray. Hybridization was performed under
LifterSlip (Erie Scientific Company, Portsmouth, NH, USA),
which allows for even dispersal of hybridization solutions
between the microarray and coverslip. The hybridization
chamber was laid on a Three-phase Tiling Agitator
(CapitalBio Corp.) to prompt the microfluidic circulation under the
coverslip. The array was hybridized at 42C overnight and
washed with two consecutive washing solutions (0.2% SDS,
2 SSC for 5 minutes at 42C and 0.2% SSC for 5 minutes at
Arrays were scanned with a confocal LuxScan scanner
(CapitalBio Corp.) and the data of obtained images were
extracted with SpotData software (CapitalBio Corp). In order
that the aCGH results were also represented with the
fluorescence intensity ratio, a spatial and
intensity-dependent normalization based on a LOWESS program was
employed, which is prevalent in microarray expression
profiling . Since each gene was represented in triplicate on each
slide and the experiments were performed in duplicate by dye
swap, producing six data points, the average ratio (always
sample/reference strain 8004) of each gene was input into
hierarchical clustering with an average linkage algorithm for
All the aCGH data can be accessed at the National Center for
Biotechnology Information Gene Expression Omnibus (GEO)
database  with accession number GSE5087.
Putative AHD CDSs identified by aCGH were examined by
PCR using the primers designed within CDSs in strain 8004.
The oligonucleotide primers and the PCR results are shown in
Additional data file 3.
Whole genome comparison of the CDS set of strain 8004 with
that of each sequenced xanthomonad strain was carried out
using the BLASTN program . Shared genes were defined
using an e-value cutoff of e-20. The CDS sets were obtained
from GenBank with the following accession numbers (in
parentheses): Xcc 8004 (CP000050), Xcc ATCC33913
(AE008922), X. axonopodis pv. citri (Xac) 306 and plasmids
pXAC33 and pXAC64 (AE008923, AE008924 and
AE008925, respectively), X. oryzae pv. oryzae (Xoo)
KACC10331 (AE013598), Xoo MAFF311018 (NC_007705),
X. campestris pv. vesicatoria (Xcv) strain 85-10 and its four
plasmids pXCV2, pXCV19, pXCV38, and pXCV183
(AM039948, AM039949, AM039950, AM039951, and
The phylogenetic relationships of all the Xcc strains tested
and other xanthomonad strains used as references were
constructed by the maximal parsimony method based on
pairwise comparisons of partial 16S-23S rDNA ITSs, which were
obtained from direct ITS rDNA sequencing of Chinese strains
and from GenBank with the accession number of each
xanthomonad strain: X. axonopodis pv. aurantifolii (Xaa) strain
X84 (AF442739.1), Xcc strain XCC15 (AF123092.2), X.
axonopodis pv. dieffenbachiae (Xad) ATCC23379 (AY576642.1),
Xad X195 (AY576648.1), X. arboricola pv. pruni (Xap)
(AJ936965.1), X. gardneri (Xg) strain CNPH496
(AY288083.1), X. vesicatoria (Xv) strain CNPH345
(AY288080.1), Xv XV1111 (AF123088.2), and other strains,
such as Xcc 8004, Xcc ATCC33913, Xac 306, Xcv 85-10, Xoo
KACC10331, and Xoo MAFF311018 with the same accession
number as that for each genome.
The genomic dissimilarity * values (the average dinucleotide
relative abundance difference) between the putative variable
genomic region in Xcc (XVR) and the genome sequence of
Xcc strain 8004 were determined by the -WEB program
[55,92] and are listed in Table 5. A BLASTN search in
GenBank was carried out for each XVR in order to identify the
origin of potential horizontal gene transfer if the homology was
To identify the genetic determinants for host specificity of
Xcc, a correlation analysis was performed using the CORREL
tool in Excel (Microsoft Office 2000). Prior to statistical
operation, the aCGH result of each gene in any Xcc strain was
transformed from the ratio value to the numerical code: 0 =
absent or highly divergent; 1 = present. The pathogenicity test
results were transformed from a qualitative description to a
numerical code: 0 = non-pathogenic; 1 = pathogenic. The HR
results were transformed from a qualitative description to a
numerical code: 0 = no HR; 1 = HR. For one round of
statistical operation, a direct correlation analysis between virulence
scales of the 20 Xcc strains (including 18 Chinese stains,
strain ATCC33913 and strain 8004) on one given plant
cultivar and the distribution pattern of each gene in 20 Xcc
strains was carried out using the program CORREL. Twelve
operations were performed and the CC values of each
operation were listed in one column, parallel to the gene list of
strain 8004 (Additional data files 8 and 9).
In each correlation analysis (for each plant assay), the Xcc
genes with the maximal R absolute values were selected as the
candidates responsible for host specificity of Xcc strain 8004
on a particular plant. Due to the possibility of more than one
gene having the same distribution pattern among 20 Xcc
strains, more than one candidate gene for each genetic
determinant was able to be selected (Table 10).
Construction of the T4SS-deletion mutant of Xcc
The virB/D4 T4SS deletion mutant was generated by the
marker exchange method. The upstream and downstream
fragments flanking the virB/D4 cluster were amplified with
the primer sets DT4-LF/DT4-LR (Additional data file 1) (the
coordinate position of the amplified fragment in Xcc 8004
chromosome is from 1956072 to 1957097, and
DT4-RF/DT4RR (the coordinate position of the amplified fragment in Xcc
8004 chromosome is from 1965913 to 1966832, respectively).
Simultaneously, the gentamicin resistant fragment was
amplified with the primer sets Gm-F/Gm-R (Additional data
file 1). The obtained fragments were cloned into the
EcoRIKpnI-BamHI-XbaI sites of the suicide vector pK18mobsacB
 one by one, yielding the recombinant plasmid pKDT4.
The plasmid pKDT4 was transferred into Xcc wild-type strain
8004 by triparent conjugation and kanamycin resistant
transconjugant colonies were screened. Bacterial cells
cultured in NYG broth without antibiotics overnight from a
single transconjugant colony chosen randomly were diluted
gradiently and plated on the NYG agar plats with 5% sucrose
and appropriate gentamicin. The gentamicin resistant and
kanamycin sensitive colonies were screened, generating the
deletion mutant of virB/D4 T4SS, named 8004T4 (Figure
5). The deletion mutant 8004T4 was further confirmed by
PCR with the primer sets DT4-LF/DT4-RR (Additional data
file 1) and the primer sets of each ORF of virB/D4 T4SS
(Additional data file 7). The virulence of the mutant was
tested on host plants by the leaf-clipping inoculation method
Functional analysis of genetic determinants for host
The candidate avr genes (XC2602, XC2004 and XC2081) of
Xcc 8004 were disrupted by using the plasmid pK18mob, a
conjugative suicide plasmid in Xcc . The internal
fragment of each target gene was amplified by PCR using
chromosomal DNA of Xcc strain 8004 as template and the primers
designed according to certain CDSs (Additional data file 1),
and cloned into the plasmid pK18mob to generate a
recombinant plasmid. The identity of the cloned fragment was
confirmed by sequencing. Each recombinant plasmid was
transformed into Escherichia coli JM109 (Additional data file
1) and then introduced into the wild-type strain 8004 by
triparental conjugation using the helper plasmid pRK2073
(Additional data file 1). Transconjugants were selected on the
NYG plates containing rifampicin and kanamycin. Mutants
were screened for disruption of the target gene by PCR using
primer PMOB-SP (Additional data file 1), a specific primer
from pK18mob, and a specific primer of the upstream gene of
each target gene (Additional data file 1). The obtained
mutants of XC2004, XC2602 and XC2081 were named
NK2004, NK2602 and NK2081, respectively.
The complementation of the mutation of each target gene was
carried out by introduction of the broad host range cosmid
pLAFR3 carrying the intact target gene into the
corresponding mutant strain. The intact target gene was
amplified by PCR using chromosomal DNA of Xcc 8004 as
template and the specific primer sets (Additional data file 1),
and cloned into the plasmid pLAFR3 under the control of the
Plac promoter. The identity of the cloned target gene was
confirmed by sequencing. The confirmed recombinant plasmid
was transformed into E. coli JM109 and then introduced into
the corresponding mutant strain by triparental conjugation.
The transconjugants were screened on NYG plates with
rifampicin, kanamycin and tetracycline. The created
complementary strains for the mutants NK2602, NK2004 and
NK2081 were named CNK2602, CNK2004 and CNK2081,
For verification of the avr function of putative avrXccE1, the
plasmid containing XC2602 was transferred into the Chinese
strains CN01, CN05, CN10 and CN11, which contain no
homologs of XC2602. A 1,605 bp fragment that includes the
region from 514 bp upstream of the star codon to 29 bp
downstream of the stop codon of XC2602 was amplified with the
primer set XC2602CM-F/XC2602CM-R (Additional data file
1) using the total DNA of Xcc 8004 as template. After
confirmation by sequencing, the fragment was cloned into the
promoterless cosmid pLAFR6 to generate the recombinant
plasmid named pC2602. The recombinant plasmid pC2602
was transferred into the strains CN01, CN05, CN10 and CN11
by triparental conjugation. The transconjugants carrying
pC2602 were screened on NYG plates with rifampicin and
tetracycline, and named CN01/pC2602, CN05/pC2602, CN10/
pC2602 and CN11/pC2602, respectively. The virulence of the
obtained strains CN01/pC2602, CN05/pC2602, CN10/
pC2602 and CN11/pC2602 on Chinese cabbage cv.
Zhongbai83 was tested by the leaf-clipping method described above.
aCGH, array-based comparative genome hybridization;
AHD, absent/highly divergent; CC, correlation coefficient;
CDS, coding sequences; cv., cultivar; HR, hypersensitive
response; ITS, intergenic spacer; LPS, lipopolysaccharide;
ORF, open reading frame; T4SS, type IV secretion system;
Xcc, Xanthomonas campestris pathovar campestris; XVR,
Xanthomonas variable genomic region.
JLT and YQH were responsible for strategic planning and
managing the overall project. LZ, BLJ, JC, and XXL
constructed the microarray and performed the aCGH
analyses. RQX, SSZ, GTL and JQ performed the isolation and
characterization of the Chinese Xcc strains. BLJ, RQX, ZCZ, MLW
and JXF constructed the mutants of the putative avr genes
and the T4SS deletion mutant. DJT, JRC, XZ and JL
performed plant assays. LZ, WJ and YQH performed the
bioinformatic analysis. JLT, YQH and BC performed CC and
other data analyses. JLT, YQH and LZ wrote the paper. All
authors have read and approved the final manuscript.
Additional data files
The following additional data are available with the online
version of this paper. Additional data file 1 contains Tables S1
and S2, which summarize the bacterial strains and plasmids
and the primers used in this study, respectively. Additional
data file 2 is a figure showing a maximal parsimony
dendrogram depicting phylogenetic relationships of partial 16S-23S
rDNA ITS sequences of all of the Chinese Xcc strains
examined and other Xanthomonas spp. Additional data file 3 is a
figure illustrating the confirmation of some present or AHD
genes defined by aCGH. Additional data file 4 is a table
presenting detailed data on the aCGH results. Additional data file
5 is a table showing the re-annotation of genes from XC2070
to XC2086 in the genome of Xcc strain 8004. Additional data
file 6 is a table listing the 305 proven/presumed
pathogenicity genes among Xcc strains revealed by aCGH analyses.
Additional data file 7 is a figure showing the deletion and
confirmation of the T4SS locus in Xcc 8004. Additional data file
8 contains the numerical codes transferred from the results of
aCGH analyses and plant tests. Additional data file 9 is a table
presenting the coefficient values of correlation between plant
test results and the gene distribution patterns of Xcc strains.
We are grateful to Dr J Maxwell Dow, Dr Robert Ryan, and Dr Ou Hongyu
for their helpful discussions and suggestions, to Professor Matthieu Arlat
for pepper seeds. We thank Dr Feng Jie for isolating some Chinese Xcc
strains. This work was supported by the '973' Program of the Ministry of
Science and Technology of China (2006CB101902), the '863' Program of
the Ministry of Science and Technology of China (20060102Z1097 and
2006AA10Z185), and the National Science Foundation of China
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