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Journal of Bacteriology, April 2000, p. 1844-1853, Vol. 182, No. 7
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of Two Novel
hrp-Associated Genes in the hrp Gene Cluster of
Xanthomonas oryzae pv. oryzae
Weiguang
Zhu,
Mark M.
MaGbanua,§ and
Frank F.
White*
Department of Plant Pathology, Kansas State
University, Manhattan, Kansas
Received 15 June 1999/Accepted 3 January 2000
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ABSTRACT |
We have cloned a hrp gene cluster from
Xanthomonas oryzae pv. oryzae. Bacteria with mutations in
the hrp region have reduced growth in rice leaves and lose
the ability to elicit a hypersensitive response (HR) on the appropriate
resistant cultivars of rice and the nonhost plant tomato. A 12,165-bp
portion of nucleotide sequence from the presumed left end and extending
through the hrpB operon was determined. The region was most
similar to hrp genes from Xanthomonas
campestris pv. vesicatoria and Ralstonia
solanacearum. Two new hrp-associated loci, named
hpa1 and hpa2, were located beyond the
hrpA operon. The hpa1 gene encoded a 13-kDa
glycine-rich protein with a composition similar to those of harpins and
PopA. The product of hpa2 was similar to lysozyme-like
proteins. Perfect PIP boxes were present in the hrpB and
hpa1 operons, while a variant PIP box was located upstream
of hpa2. A strain with a deletion encompassing
hpa1 and hpa2 had reduced pathogenicity and
elicited a weak HR on nonhost and resistant host plants. Experiments
using single mutations in hpa1 and hpa2
indicated that the loss of hpa1 was the principal cause of
the reduced pathogenicity of the deletion strain. A 1,519-bp insertion
element was located immediately downstream of hpa2.
Hybridization with hpa2 indicated that the gene was present in all of the strains of Xanthomonas examined.
Hybridization experiments with hpa1 and IS1114
indicated that these sequences were detectable in all strains of
X. oryzae pv. oryzae and some other Xanthomonas species.
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INTRODUCTION |
The hrp ("harp")
genes encode type III secretory pathways and are required by many
phytopathogenic bacteria to elicit a hypersensitive response (HR) on
nonhost or resistant host plants and for pathogenesis on susceptible
hosts. The HR is a rapid localized death of the host cells that occurs
upon pathogen infection and, together with the expression of a complex
array of defense-related genes, is a component of plant
resistance. The hrp genes were first identified in
Pseudomonas syringae pv. phaseolicola, a bean pathogen
(38). Since then, hrp genes from a variety of
plant pathogenic bacteria, including Erwinia,
Pseudomonas, Ralstonia, and
Xanthomonas, have been characterized (for reviews, see
references 2, 9, and 11). The
specific functions of the hrp pathway in pathogenesis are
not known. However, type III secretion pathways of animal and plant
pathogens have been demonstrated to mediate the secretion of virulence
factors into the extracellular melieu. Some of the proteins ultimately
end up in the host cell cytoplasm (reviewed in references 11,
25, and 37). In mediating the interaction of the bacterium and the host plant, the hrp pathway
presumably acts to prevent or inhibit a general resistance response or
otherwise enhance the colonization of the plant by the bacteria.
Given the importance of the type III systems to pathogenicity, it can
be expected that analysis of the systems in different species will
provide insight into the adaptation of the species to their respective
host plants. The hrp gene clusters appear to group into two
types on the basis of sequence relatedness and operon organization
(reviewed in reference 9). The hrp genes of Pseudomonas and Erwinia comprise one group,
and the hrp genes of Xanthomonas campestris pv.
vesicatoria and Ralstonia solanacearum comprise the second
group. Our present understanding of the group 2 hrp genes is
based almost entirely on the characterization of two strains
representing R. solanacearum and X. campestris
pv. vesicatoria. The genus Xanthomonas itself is comprised
of a large number of different species and pathovars that colonize over
392 species of plants (36). Xanthomonas oryzae
pv. oryzae is the causal pathogen of bacterial leaf blight on rice
(54). We report here the cloning of a hrp cluster
from X. oryzae pv. oryzae and sequence analysis of the left
end of the region. In the process of characterization, we identified
two novel loci that are associated with the hrp cluster and
an insertion sequence (IS) element not previously characterized in
X. oryzae pv. oryzae.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The bacterial strains and
plasmids used in the experiments are listed in Table
1. Escherichia coli strains
were grown in Terrific broth (TB) or on Luria agar plates at 37°C
with the appropriate antibiotics. X. oryzae pv. oryzae
strains were cultured on tryptone sucrose agar or in TB at 28°C. The
genomic library of PXO86 in pHM1 was described previously
(24). Carbenicillin, spectinomycin, and kanamycin were used
at 100 µg/ml.
Recombinant DNA techniques.
DNA manipulations were performed
by standard procedures (6). Restriction enzymes, T4 DNA
ligase, Klenow fragment of DNA polymerase I, and Taq
polymerase were purchased from Life Technologies, Inc. (Gaithersburg,
Md.) and Fisher Scientific (St. Louis, Mo.). Chemicals were purchased
from Sigma Chemical (St. Louis, Mo.) and Fisher Scientific. BioTrace HP
membrane (Gelman Sciences, Ann Arbor, Mich.) was used for Southern blot
hybridization. EcoRI DNA fragments from p23-44, a cosmid
clone containing hrp genes of X. oryzae pv.
oryzae, were subcloned into pBluescript KS(+) (Stratagene, Inc., La
Jolla, Calif.). The DNA sequences of the two strands of these subclones
were determined by the DNA Sequencing Facility of Iowa State
University. Amino acid alignments were constructed using Clustal W 1.7 (55). Similarity searches were performed using the BLAST
program (4). Identity and similarity comparisons among
proteins were performed using the Fasta program (45).
Potential signal peptides at the N-terminal and transmembrane domains
of the proteins were predicted by PSORT (41, 42) and TopPred2 (60), respectively. Specific motif searches were
performed using the MOTIF program (43).
Transposon and deletion mutagenesis.
Mutagenesis of p23-44
with Tn5-gusA1 was performed in E. coli strain
DH5
MCR. Cells growing exponentially in TB were infected with
bacteriophage p
A1 carrying Tn5-gusA1 (51).
After 1 h of incubation at 37°C, which allowed the phage to be
absorbed and phenotypic expression of kanamycin resistance, bacteria
were plated on Luria agar medium supplemented with kanamycin and
incubated overnight at 28°C. Plasmid DNA was extracted from single
colonies, digested with EcoRI, and analyzed by
electrophoresis on 1% agarose gels. Each plasmid with a transposon in
the bacterial genomic DNA of the clone was transformed into
PXO99A by electroporation or by conjugation using the
conjugal helper strain S17-1 (53). Marker exchange
mutagenesis was performed using spontaneous homologous recombination
and screening for kanamycin-resistant, spectinomycin-sensitive clones.
Mutations were confirmed by Southern blot analysis of bacterial genomic
DNA using 32P-labeled probes for subclones of p23-44. Four
isolates from each marker exchange were tested on tomato and rice
plants. The p23-44 cosmid was reintroduced by electroporation into the
hrp mutants for complementation tests.
Mutations in
hpa1,
hpa2, and
hrpA were
made using GPS-1 of the Genome Priming System (New England BioLabs,
Beverly, Mass.).
GPS-1 contains a modified Tn
7 with the
nptII gene for resistance
to kanamycin, and insertions were
generated in vitro in pK4.0
and pK6.0B according to the instructions of
the manufacturer (
14).
One mutation in
hpa1 was
generated by introducing the gene for
resistance to kanamycin from
pUC4K into the
EcoRI site of
hpa1 (
58). Marker exchange mutations of
hrpA were
created by first
introducing the GPS-1 mutations into p23-44 in
E. coli. Recombinants
were generated by introduction of both
plasmids into the Rec
+ strain of
E. coli TB1.
Recombinant plasmids were rescued by electroporation
into
E. coli strain C2110 (
35), which is deficient in
polymerase
I activity and does not permit replication of ColE1
replicons
(pK4.0 or pK6.0B), and selection for resistance to
spectinomycin
and kanamycin. The resulting recombinant cosmids were
transformed
into
E. coli strain S17-1 and moved from S17-1
into the PXO99
A strain of
X. oryzae pv. oryzae
by biparental mating. Four colonies
were selected for marker exchange
mutagenesis as described
above.
The deletion in the left end of the
hrp cluster covering
hpa1 and
hpa2 was created by replacing a 2.1-kb
SalI/
XhoI fragment
in pK6.0B with 1.3-kb
SalI fragment containing a kanamycin resistance
gene from
pUC4K (
58). The resulting plasmid, pK6.0B
2, was
introduced
into p23-44 by homologous recombination in
E. coli as described
above for the GPS-1
hrpA mutations.
The resulting cosmid, p23-44
2,
was first transformed into
E. coli strain S17-1 and then moved
from S17-1 into
X. oryzae pv. oryzae PXO99
A by biparental mating. Twenty
colonies that were sensitive to
spectinomycin and resistant to
kanamycin after growth on nonselective
media were obtained and tested
for virulence and HR on rice and
tomato.
Plant assays.
Pathogenicity and hypersensitivity assays were
performed as described previously (24). Tomato cv. VFN8 was
used for the nonhost hypersensitivity test. Ten-day-old rice seedlings
IRBB10 and IRBB7, containing corresponding resistance genes
Xa10 and Xa7, respectively, were used for
race-specific resistance assays. IR24 was used for pathogenicity
testing. All plants were grown in growth chambers at 28°C (daytime)
and 25°C (nighttime) with a 14-h photoperiod and 85% humidity. For
hrp phenotype assays, inoculum concentrations were adjusted
to an optical density at 600 nm of 1.0 (approximately 2 × 109 CFU/ml) using a DU-64 spectrophotometer (Beckman
Instruments). Inoculum concentrations for hpa1 and
hpa2 mutation phenotype assays were adjusted to an optical
density at 600 nm of 0.3. The differences between wild-type and mutant
pathogenicity and hypersensitivity reactions were enhanced at the lower
dilution. Growth of bacteria on rice after infiltration was monitored
as previously described (24).
Sequence analysis of PXO99A.
A 651-bp region was
amplified by PCR from PXO99A using the primers
5'-GATTGTCTGCGGAAAATAG-3' (IS99FOR) and
5'-GGTACGCAGCAGATCTGGG-3' (IS99REV), cloned into
pCR2.1-TOPO (52), and sequenced. The parameters used
for PCR were as follows: step 1, 95°C for 2 min; step 2, 50°C for
30 s; step 3, 72°C for 90 s; step 4, 95°C for 30 s;
step 5, 35 cycles from step 2 to step 4; step 6, 72°C for 2 min.
Southern hybridization analysis.
Total DNA isolation and
Southern hybridization analysis were as previously described
(34). Probes were prepared by either PCR (49) or
gel purification and random priming (6). The 408-bp
EcoRI/XhoI fragment from hpa1 was used
as a probe for hpa1-related sequences. A 321-bp probe for
hpa1 was generated by PCR using internal primers
5'-AACAGGATCCAGATTGCTTCGAAGAGGCTGCC-3' (HPA2F1) and
5'-AACAAGGATCCGCATATTTATCACGCTCC-3' (HPA2R1). A 1522 base pair probe for IS1114 was amplified using primers
5'-AGTCGCCCCTGAAAAACCCCCAG-3' (ISHRPF1) and
5'-AAGTCGCCCCTGAAAAACCCTC-3' (ISHRPR1). PCR conditions for
both probes were as described above. Probes were labeled using a
Rediprime random-priming labeling kit (Amersham, Arlington Heights, Ill.).
Nucleotide sequence accession numbers.
The DNA sequence for
the hpa2-to-hrpB region has GenBank accession no.
AF232057. The sequence for the region including IS1114 from
PXO86 is under GenBank accession no. AF232058. The sequence of the
corresponding region of IS1114 insertion in PXO99A is under GenBank accession no. AF232714.
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RESULTS |
Isolation of the hrp region of X. oryzae
pv. oryzae.
Cosmids p23-14, p23-44, and p2-2 were recovered from a
genomic library of strain PXO86 by using p83-15, which contained a portion of the X. campestris pv. vesicatoria hrp
region (10), as a probe. The maps of the clones were
characterized by endonuclease restriction digests, Southern blotting,
and DNA sequence analysis and shown to cover the regions corresponding
to hrpA, hrpB, hrpC, and
hrpD of X. campestris pv. vesicatoria (Fig.
1). Thirty-five strains with
Tn5-gusA1 insertions in the region covered by p23-44 were
generated from strain PXO99A of X. oryzae pv.
oryzae. Eleven strains, with insertions in the 4.8- and 7.5-kb
EcoRI fragments, had unaltered hrp gene function. Twenty-four strains, with insertions in the 8.3-, 1.4-, and 5.1-kb EcoRI fragments, lost the ability to elicit disease on rice
and an HR on tomato (Fig. 1). The mapping data indicated that the insertions in hrp loci were located in the hrpB,
hrpC, and hrpD operons. None of the
Tn5-gusA1 insertions appeared to have inserted into the
hrpA operon. Therefore, two insertions in hrpA
were generated in pK4.0 by using pGPS-1 and recombined into the
hrp region (Fig. 1, insertions 3230 and 2785). Both
mutations resulted in hrp mutant phenotypes. The phenotypes
of a representative hrp mutant after inoculation on rice and
tomato are shown in Fig. 2A. Introduction of p23-44 into selected hrp mutants restored the ability to
elicit disease on rice and an HR on tomato (Fig. 2A). In planta growth of a representative hrp mutant was reduced compared to that
of the wild-type strain, while the levels of in planta growth of the
mutant strains carrying p23-44 were similar to that of the parent
strain PXO99A (Fig. 2B).
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FIG. 1.
Restriction fragment map of the hrp region in
X. oryzae pv. oryzae. The name of each fragment also
indicates the size in kilobases. Vertical lines above the map indicate
positions of Tn5-gusA1 insertion. Lines with filled circles
indicate that the insertion abolished hrp function; lines
without circles indicate insertions that did not cause loss of
hrp activity. Arrows indicate positions and orientations of
hrp operons. E, EcoRI; K, KpnI.
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FIG. 2.
Effects of mutations in the hrp genes of
X. oryzae pv. oryzae. (A) Phenotype of rice (cultivar IR24)
and tomato leaf (VFN8) reactions to the following strains: 1, PXO99A; 2, PXO99A9mx (hrpC); 3, PXO99A9mx (p23-44). Water soaking and disease symptoms were
evidenced by discoloration at the inoculation site on rice leaves
(left). Inoculation in the hrp mutants resulted in no change
in leaf coloration (leaf 2). HR on tomato leaf (right) is indicated by
light gray patches (leaves 1 and 3). (B) Effect of hrp
mutation on growth of bacteria in rice leaves. Numbering as for panel
A.
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X. oryzae pv. oryzae interacts with rice plants in a
race-specific manner. PXO99
A with
avrXa10 or
avrXa7 elicits an HR on rice cultivars with the
corresponding resistance gene
Xa10 or
Xa7
(
24). Three
hrp mutants
of
X. oryzae
pv. oryzae, PXO99
A8mx, PXO99
A9mx, and
PXO99
A56mx, whose insertions could be mapped in the
hrpB,
hrpC, and
hrpD operons,
respectively, were unable to elicit a race-specific
HR when carrying
avrXa10 (Table
2). Identical
results were obtained
with
avrXa7 (data not shown).
Two novel hrp-associated loci are located in the left
end of the hrp cluster.
The DNA sequence was
determined for 12,165 bp from the BglI site in the 7.5-kb
EcoRI fragment (E7.5) and extending 713 bp into the 1.4-kb
EcoRI fragment of p23-44 (Fig.
3). The sequence data were organized into
two portions. The first portion, starting at the second SalI
site in E7.5, contained 10,096 bp and included transcription units A
and B and two additional open reading frames, tentatively termed
hpa1 and hpa2 (Fig. 3). Table
3 gives the positions and properties of
the noted features in the first portion. The second portion, containing
2,075 bp and the IS element IS1114, extended upstream from
the SalI site to a BglII site in the E7.5 fragment (Fig. 3).
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FIG. 3.
Region of sequence analysis of the left hrp
region of PXO86. Transcriptional units and open reading frames of the
hrp genes are indicated by lines and open arrows,
respectively. The direction of transcription and translation is
indicated by direction of the arrow. Filled bars indicate sequenced
regions. E, EcoRI; K, KpnI.
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The
hrpA operon contained a single coding sequence for HrpA
(HrcC, using the unified nomenclature [
8]), which
started at
position 4123 and continued to position 2306. The sequence
predicted
a protein of 605 amino acids with 96% similarity to HrpA1 of
X. campestris pv. vesicatoria (
63). HrpA from
X. oryzae pv. oryzae
lacked two alanine residues
corresponding to positions 295 and
296 in HrpA1 from
X. campestris pv. vesicatoria (
63) and was,
therefore, two
amino acids shorter than HrpA1 from
X. campestris pv.
vesicatoria. The
hrpB locus of
X. oryzae pv.
oryzae contained
eight coding regions extending from position 9820, which is the
start of HrpB1, to the end of HrpB8 at 4211. The putative
GTG
start codon for HrpB1 was based on the alignment to HrpB1 of
X. campestris pv. vesicatoria, which has the usual ATG start
codon
(
18). A PIP box (PIP-3), which consists of two direct
repeats
(TTCGC) separated by 15 nucleotides, was located 81 bp upstream
of the putative HrpB1-coding sequence and was similar to the PIP
box
found in
X. campestris pv. vesicatoria (
18) and
R. solanacearum (
21). PIP-3 was separated by 14 nucleotides from another PIP
box (PIP-4) in the opposite direction.
PIP-4 was located 155 nucleotides
away from the start codon of the
putative HrpC1-coding sequence.
(Complete sequence analysis of the
hrpC operon will be presented
later.)
Sequence analysis at the left end of the 8.3-kb
EcoRI
fragment revealed an open reading frame for a protein of 143 amino acid
residues, starting at position 1136, that had not previously been
described for
Xanthomonas and was tentatively named
hpa1 (Fig.
4). A perfect
consensus PIP box was located at position 975 and
135 bp upstream of
hpa1. The putative protein encoded by
hpa1 is
glycine rich (26% glycine), particularly in the middle and
C-terminal
portions, and has no high degree of sequence similarity
to proteins in
the databases.
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FIG. 4.
DNA sequence of hpa1. Sequences for a PIP
box, restriction sites, and a ribosome binding site (SD) are
underlined. The sequence of the deduced translation product is given in
the single-letter code below the DNA sequence. The glycine-rich regions
are double underlined. Asterisks indicate a potential transmembrane
motif.
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The
hpa2 open reading frame was oriented in the opposite
direction to
hpa1. The putative protein product has a high
degree
of similarity at the amino acid level to a group of
lysozyme-like
proteins starting at position 569 (Fig.
5A). An imperfect PIP
box (with C
replaced by a T in the second TTCGC consensus repeat)
was located
upstream of
hpa2 at position 884 and 165 bp upstream
from
the first ATG (Fig.
5B). The putative start codon for Hpa2
was unclear.
The first upstream ATG was 150 bp from the region
of similarity to
lysozyme-like proteins (Fig.
5B). A consensus
secretion signal sequence
was identified within 54 bp upstream
from the start of the sequence
similarity to lysozyme-like proteins
(Fig.
5B). The putative cleavage
site was predicted to occur immediately
before the start of the
sequence similarity.
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FIG. 5.
Sequence analysis of hpa2. (A) Sequence
alignment of Hpa2 and selected proteins. Bkpm, Burkholderia
pseudomallei (GenBank accession no. AAD05172); IagB,
Salmonella enterica serovar Typhimurium (39);
IpgF, Shigella flexneri (3); VirB1,
Agrobacterium tumefaciens (40); LyzC, chicken
(28). The conserved A and E -helices (B and E) and
 -sheet () in the structure of lysozyme are overlined. Asterisks
indicate conserved asparagine and aspartate residues in the catalytic
region of lysozyme. Gaps in the alignment are represented by dashes.
Hpa2 is shown as starting from the isoleucine that is 16 residues
upstream of the putative cleavage site. The triangle indicates the
putative cleavage site. (B) Promoter region of hpa2. A
HindIII site and imperfect PIP box (PIP-1) are
underlined. Only amino acids starting from the last methionine before
the conserved region are indicated. The predicted signal peptide in the
protein product is underlined.
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An IS element is located adjacent to hpa2 in
PXO86.
An IS element was identified 254 bp from the end of
hpa2. The element is 1,519 bp long and bounded by an 18-bp
perfect inverted repeat. IS1114 was similar in sequence to
two recently described insertion elements from Rhizobium and
X. campestris pv. campestris (Fig.
6A) (13, 50). The repeat was
located within a 6-bp direct repeat (TAAAA) that may have been
generated by the insertion process (Fig. 6B). Strain PXO99A
did not contain IS1114 adjacent to hpa2, and the
sequence analysis of the same region revealed only the TAAAA sequence
with no duplication (Fig. 6B). The facts that the TAAAA sequence, which
is duplicated at the ends of IS1114 in PXO86, was not
duplicated in PXO99A and that no remnant of
IS1114 was found at the corresponding region of
PXO99A suggest that the insertion is unique to the PXO86
lineage. The sequence from the BglII site to the end of
hpa2 without the IS element did not match any entries in the
GenBank database. Therefore, the element also does not appear to have
inserted into an identifiable genetic locus. Genetic loci that are
involved in pathogenicity have been associated with transmissible
genetic elements (30). These so-called pathogenicity islands
may also have G+C compositions that differ from the G+C content of the
bulk of the chromosomal DNA, reflecting the transfer among different
species of bacteria (reviewed in reference 22).
However, no evidence for a dramatic shift in the G+C content at the
element's boundary with the hrp region was observed. We
therefore could not find that the element had any relevance to
hrp function. We believe that IS1114 is present near the hrp region in some lineages and probably inserted
into the region by chance.
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FIG. 6.
Sequence analysis of IS1114. (A) Sequence
similarity of IS1114 to insertion elements
IS1478a (13) and ISRm220-13-5
(50). The alignment shows only 43 to 51 bases of sequence
from one end, with the overall sequence identity of the entire element
to IS1114 given in parentheses. The arrow indicates an 18-bp
repeat. Identities in two of the three elements are shaded. (B)
Sequence alignment of genomic DNA from strain PXO99A with
the region of insertion in strain PXO86. Only the 18-bp direct repeats
are shown (boldface). Dashes indicate spaces introduced for optimal
alignment. A direct 6-bp repeat at the site of insertion in PXO86 is
underlined.
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Deletion of the left end affects virulence.
A deletion mutant
(PXO99A2) covering the hpa1-hpa2 region was
constructed by replacing the region between the SalI and
XhoI sites of pK6.0B with the gene for resistance to
kanamycin and subsequent recombination into the chromosome of
PXO99A to give strain PXO99A2
(58). PXO99A2 was therefore missing
hpa2 entirely, and hpa1 was truncated. Upon
inoculation to rice, PXO99A2 was found to cause reduced
disease symptoms and, when harboring avrXa10, elicited a
weak HR on rice plants with resistance gene Xa10 in terms of
the intensity of browning (Fig. 7A). The
reduced disease symptoms were accompanied by reduced bacterial
populations in the leaf tissue (Fig. 7B). Similarly, the HR of the
mutant on tomato was delayed and weaker in terms of the area that
collapsed after inoculation (not shown). The reduced pathogenicity of
the mutant could be complemented by reintroduction of p23-44 (Fig. 7B).
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FIG. 7.
Effect of an hpa1-hpa2 deletion on virulence
and avrXa10 activity. (A) Phenotypes of
PXO99A2 and PXO99A2(pFWX10-F2), which
contains the avirulence gene avrXa10, on susceptible (IR24)
and resistant (BB10) rice cultivars. Susceptible leaves (upper two
leaves) and resistant leaves (lower two leaves) were photographed 3 days after inoculation with the strain indicated at the right. (B)
Growth of PXO99A2 in susceptible rice cultivar IR24. 1, PXO99A; 2, PXO99A2; 3, PXO99A2(p23-44).
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Insertions and one deletion were generated in pK6.0B, which by itself
restored full pathogenicity to PXO99
A2, and the mutants
were tested for the ability to restore pathogenicity
(Fig.
8). The insertions in
hpa2
(pK6.0B-479) and immediately
downstream from
hpa1
(pK6.0B-1795) did not affect the ability
of pK6.0B to restore water
soaking to PXO99
A2 (Table
4). Plasmids pK6.0B-1116 and pK6.0B-1139,
on the other
hand, failed to restore water soaking (Table
4). The
insertion
in pK6.0B-1116 was located 20 bp upstream of the
hpa1 start codon
and interrupted the presumed promoter
elements from the coding
sequence of
hpa1. The plasmid
pK6.0B-1139 had an insertion at
the
EcoRI site in the coding
sequence of
hpa1. Thus, plasmids
with
hpa1
mutations were unable to restore water soaking to
PXO99
A2, indicating that the loss of
hpa1
was the principal cause of
the reduced virulence of
PXO99
A2.
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FIG. 8.
Map of single-gene mutations in hpa1 and
hpa2. Insertion sites are indicated by lines with circles at
the top. Filled circles indicate no loss of virulence; open circles
indicate insertions that reduced virulence. The filled bar indicates
the region missing in deletion 2. IS1114 is indicated by
the hatched bar. B, BglII; E, EcoRI; H,
HindIII; K, KpnI; S, SalI; X,
XhoI.
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Distribution of hpa1, hpa2, and
IS1114 in species of Xanthomonas.
To determine
the prevalence of the newly identified elements in a variety of other
strains of X. oryzae pv. oryzae and X. campestris, gene-specific and element-specific probes were
generated from hpa1, hpa2, and IS1114
by PCR or gel purification of an internal restriction fragment. The
probes were then used in Southern analyses of genomic DNA. Both
hpa1 and IS1114 hybridized to DNAs from all of
the strains of X. oryzae pv. oryzae and the one strain of
X. oryzae pv. oryzicola that were tested (Fig. 9A and
B, lanes 1 to 5). Signal for
hpa1 was detected in DNA from X. campestris pv.
alfalfae KX-1, X. campestris pv. malvacearum H, and X. campestris pv. phaesoli SC-4A and was not detected in X. campestris pv. vesicatoria 81-23, X. campestris pv.
campestris KXXC-1, or X. campestris pv. holcicola (Fig. 9A,
lanes 6 to 11). Outside X. oryzae, IS1114 was
detected only in X. campestris pv. campestris KXXC-1 and
X. campestris pv. malvacearum H (Fig. 9B, lanes 7 and 10).
In contrast to hpa1 and IS1114, hpa2
was detected in DNAs from all of the tested strains (Fig. 9C).
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|
FIG. 9.
Southern analysis of hpa1, hpa2,
and IS1114. (A) DNA probed with the
EcoRI/XhoI fragment of hpa1. (B) DNA
probed with the PCR fragment from IS1114. (C) DNA probed
with the PCR fragment from hpa2. Genomic DNA was extracted
from 11 strains of Xanthomonas spp. and digested with
EcoRI (A and B) or SalI (C). Lanes 1, X. oryzae pv. oryzae PXO86; lanes 2, X. oryzae pv. oryzae PXO99A; lanes 3; X. oryzae pv. oryzae PXO177; lanes 4, X. oryzae pv.
oryzae C191; lanes 5, X. oryzae pv. oryzicola BLS 303; lanes
6, X. campestris pv. vesicatoria 81-23; lanes 7, X. campestris pv. campestris Kxxc1; lanes 8, X. campestris
pv. alfalfae KX-1; lanes 9, X. campestris pv. holcicola;
lanes 10, X. campestris pv. malvacearum H; lanes 11, X. campestris pv. phaesoli SC-4A. See Materials and Methods
for PCR primers and conditions. Numbers at the left indicate kilobase
pairs.
|
|
| |
DISCUSSION |
The results presented here demonstrate that a type III secretory
system, also known as the hrp system, has a critical role for pathogenicity of X. oryzae pv. oryzae on rice. Type III
secretory systems play central roles in the ability of many
gram-negative bacteria to colonize plant and animal hosts. In general,
the systems are envisaged to direct the assembly of a supramolecular
secretory apparatus similar in structure to the flagellar apparatus,
which is also the product of a type III secretory system. Some
components of the type III systems are conserved due to the structural
requirements, while other components can be expected to reflect the
adaption of the system to the particular niche of the bacterium
(26). The hrpA and hrpB operons
represent part of the conserved core of genes necessary for the
assembly of the hrp system. The predicted proteins of the
hrpA and hrpB operons had 94% or higher amino acid residue identity with the proteins from X. campestris
pv. vesicatoria (10, 57). HrpA is a member of the HrcC class
of hrp proteins and is localized to the outer membrane,
where it is thought to function the transporter past the outer membrane layer (63). As a class, HrpA proteins have similarity to a
variety of proteins from other type III systems, and in fact, HrpA
ancestry can be traced to other secretory systems (63).
HrpB3, HrpB5, HrpB6, and HrpB8 of the hrpB operon are
related to components of other type III systems, including the
flagellar assembly pathway (8). HrpB8 and HrpB3, for
example, are similar in amino acid sequence to FliR and FliF, which
have been determined to be components of the inner membrane basal body
and M-ring portion of the flagellar secretory apparatus, respectively
(16, 27). HrpB1, HrpB2, HrpB4, and HrpB7 are proteins that,
on the basis of sequence similarity, are unique to
Xanthomonas and R. solanacearum. These proteins
either have diverged from the ancestral secretion system or represent
unique adaptations of the type III system in Xanthomonas and
R. solanacearum. Divergence between the latter four proteins
in X. oryzae pv. oryzae and X. campestris pv.
vesicatoria was no greater than that for proteins with recognizable
counterparts in other type III systems, and they therefore appear not
to have diverged along species lines as a possible consequence of
adaptation to the particular host plants.
The hrp mutations in X. oryzae pv. oryzae
resulted in the loss of pathogenicity and prevented the elicitation of
the nonhost HR on tomato and the race-specific HR on incompatible rice
plants due to the avirulence genes avrXa10 and
avrXa7, which are homologs of avrBss3 from
X. campestris pv. vesicatoria (24). Activity due
to avrBs3 was also lost when X. campestris pv.
vesicatoria was hrp deficient (32), and
dependence on hrp function for avirulence activity could be
bypassed when avrBs3 and other members of the family from
X. campestris pv. malvacearum were expressed in the plant
cells (15, 56). In a separate study, we have observed a 75%
reduction in the number of transformation foci after particle bombardment of resistant rice leaves with a plant-expressed copy of
avrXa10 (66). Therefore, the protein products of
avrXa10 and avrXa7 along with possible virulence
factors are likely to be secreted by a hrp-encoded type III
secretory apparatus into the cells of the rice plant.
Despite the high degree of similarity, the analysis of the left end of
the hrp region of X. oryzae pv. oryzae revealed
two genes, named hpa1 and hpa2, that were not
found in X. campestris pv. vesicatoria. The putative
hpa1 product has an amino acid composition similar to the
compositions of the harpin proteins of P. syringae pathovars
and Erwinia species and the harpin-like PopA protein of
R. solanacearum (5, 20, 23, 61). These proteins
share regions of high glycine content and are secreted via the type III
pathway. Whether Hpa1 is secreted is unknown at present. Conditions for
hrp-dependent secretion by X. campestris pv.
vesicatoria have recently been determined (48). Thus, it may
be possible to adapt the conditions to X. oryzae pv. oryzae
and determine if the hpa1 product is secreted in a
hrp-dependent manner. Like popA, hpa1 has a PIP box immediately upstream of the coding sequence and is likely
to be regulated by the hrpXo gene product, which is a member
of the AraC class of transcription factors (29, 44, 62).
Harpins gained attention by the fact that some harpins and PopA can
elicit hypersensitive reactions on certain plants simply by injection
of the proteins into the leaf tissue (5, 23, 46, 61). The
protein product of hrpW has similarity to both harpin and
pectate lyase (12, 20, 31). However, the elicitor properties
are not shared by all harpins, and in the case of harpin from P. syringae pv. tomato, the protein elicited an HR on the host plant
(46). Mutations in hrpZ, hrpW, and
popA have no observable effect on the pathogenicity (1,
5, 12). Therefore, the biological relevance of the latter genes
or the elicitor activities of their products is unknown. On the other
hand, mutations in hrpN, from which harpins were originally
named, abolished pathogenicity of Erwinia amylovora
(61). Mutations in hpa1 reduced pathogenicity due
to X. oryzae pv. oryzae. hpa1 is the only gene
from Xanthomonas with a harpin-like product. However,
sequence similarity with the hrpW gene from P. syringae pv. tomato in the DNAs from several Xanthomonas species was recently reported (12).
Thus, Xanthomonas, like P. syringae and
Erwinia species, may produce a variety of harpin-like
proteins depending on the species or strain.
The role of the hpa2 locus, which encodes a lysozyme-like
protein, is unclear. However, an argument can be made that the locus is
another core component of the type III system. The locus appears to be
present in all of the species of Xanthomonas that were
examined, and related genes can be found in association with a variety
of secretory systems, including type III systems of animal pathogens. On the one hand, the mutation of hpa2 had no apparent effect
on pathogenicity under the conditions of the assay. Indeed, whether this protein is produced in vivo remains in question. The lack of a
phenotype with the hpa2 mutants may reflect the lack of a requirement for this gene under the conditions of testing or
environment, yet the locus may be required under natural infection
conditions. None of the lysozyme-related proteins appear to be
essential for the related systems under the conditions of testing.
Mutations in virB1 severely reduced but did not eliminate
tumor formation (40). Similarly, mutations in gene 19, which
encodes a lysozyme-like protein in the R1-16 plasmid conjugation
pathway, reduced but did not eliminate conjugative transfer of R1-16
(7). A mutation in ipgF, which is a locus in a
type III pathway of Shigella flexneri, was reported to have
no effect on pathogenicity (3). One obvious possibility for
the function of the lysozyme-like proteins would be in the degradation
of the peptidoglycan layer of the bacterial cell wall to accommodate
the respective secretory apparatuses or associated pili (19, 33,
47). This possibility remains to be tested.
| |
ACKNOWLEDGMENTS |
We thank Diana Pavlisko for assistance in the preparation of the
manuscript and Bing Yang and Nick Wills for excellent technical assistance.
This work was supported by grants 94-37303-0548 and 98-35303-6446 from
the National Competitive Research Initiative of the U.S. Department of
Agriculture. W.Z. was support in part by a grant from the Rockefeller Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 4024 Throckmorton Hall, Department of Plant Pathology, Kansas State
University, Manhattan, KS 66506. Phone: (785) 532-6176. Fax: (785)
532-5692. E-mail: fwhite{at}plantpath.ksu.edu.
Contribution number 99-500-J from the Kansas Agriculture Experiment Station.
Present address: Molecular Cardiology Division, Southwestern
Medical Center, Dallas, TX 75235-8573.
§
Present address: Dept. of Plant Pathology, Univ. of
California-Davis, Davis, CA 95616.
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Journal of Bacteriology, April 2000, p. 1844-1853, Vol. 182, No. 7
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
