Hpa2 required by HrpF to translocate Xanthomonas oryzae transcriptional activator-like effectors into rice for pathogenicity.
ABSTRACT Xanthomonas oryzae pv. oryzicola, the causative agent of bacterial leaf streak, injects a plethora of effectors through the type III secretion system (T3SS) into rice cells to cause disease. The T3SS, encoded by the hrp genes, is essential for the pathogen to elicit the hypersensitive response (HR) in nonhost tobacco and for pathogenicity in host rice. Whether or not a putative lytic transglycosylase, Hpa2, interacts with a translocon protein, HrpF, to facilitate bacterial pathogenicity remains unknown. Here we demonstrated that both the hpa2 and hrpF genes are required for the pathogenicity of X. oryzae pv. oryzicola strain RS105 in rice but not for HR induction in tobacco. The expression of hpa2 was positively regulated by HrpG and HrpD6 but not by HrpX. In vivo secretion and subcellular localization analyses confirmed that Hpa2 secretion is dependent on HpaB (a T3SS exit protein) and that Hpa2 binds to the host cell membrane. Protein-protein assays demonstrated that Hpa2 interacts with HrpF. In planta translocation of AvrXa10 indicated that the mutation in hpa2 and hrpF inhibits the injection of the HpaB-dependent transcriptional activator-like (TAL) effector into rice. These findings suggest that Hpa2 and HrpF form a complex to translocate T3S effectors into plant cells for pathogenesis in host rice.
- SourceAvailable from: nih.gov[show abstract] [hide abstract]
ABSTRACT: The Shigella flexneri invasion process requires the synthesis of the Ipa proteins and their secretion by specific factors encoded by the mxi and spa genes, which are clustered upstream from the ipa operon. We report here the characterization of the ipgD, ipgE, and ipgF genes, which are located in the 5' end of the mxi locus. Analysis of IpgF-PhoA fusions endowed with high levels of alkaline phosphatase activity confirmed the functionality of a classical signal sequence detected in the sequence of IpgF. The ipgD and ipgF genes were each inactivated on the large virulence plasmid by insertion of a nonpolar cassette; each of the ipgD and ipgF mutants thus constructed showed the same invasive phenotype as the wild-type strain and was able to provoke keratoconjunctivitis in guinea pigs. It thus appears that two genes located at the ipa-proximal part of the mxi locus are not directly involved in invasion. Analysis of concentrated culture supernatants of the wild-type and ipgD strains indicated that secretion of one polypeptide, whose size was consistent with that predicted for the IpgD protein (60 kDa), was abolished in the ipgD mutant.Infection and Immunity 06/1993; 61(5):1707-14. · 4.07 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Many Gram-negative plant and animal pathogenic bacteria use a specialized type III secretion system (TTSS) as a molecular syringe to inject effector proteins directly into the host cell. Protein translocation across the eukaryotic host cell membrane is presumably mediated by a bacterial translocon. The structure of this predicted transmembrane complex and the mechanism of transport are far from being understood. In bacterial pathogens of animals, several putative type III secretion translocon proteins (TTPs) have been identified. Interestingly, TTP sequences are not conserved among different bacterial species, however, there are structural similarities such as transmembrane segments and coiled-coil regions. Accumulating evidence suggests that TTPs are components of oligomeric protein channels that are inserted into the host cell membrane by the TTSS.Trends in Microbiology 05/2002; 10(4):186-92. · 8.43 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Pathogenicity of most Gram-negative bacterial plant pathogens depends on hrp (hypersensitive response and pathogenicity) genes, which control the ability to cause disease and to elicit specific defense responses in resistant plants. hrp genes encode a specialized type III secretion (TTS) system that mediates the vectorial delivery of bacterial effector proteins across both bacterial membranes as well as across the eukaryotic plasma membrane into the host cell cytosol. One well-studied effector protein is AvrBs3 from Xanthomonas campestris pv. vesicatoria, the causal agent of bacterial spot in pepper and tomato. AvrBs3 induces hypertrophy symptoms in susceptible plants and triggers a resistance gene-specific cell death reaction in resistant plants. Intriguingly, AvrBs3 has characteristic features of eukaryotic transcription factors, suggesting that it modulates the host's transcriptome. Here, we discuss the TTS system of X.campestris pv. vesicatoria in the light of current knowledge on type III-dependent protein secretion in plant pathogenic bacteria.The EMBO Journal 11/2002; 21(20):5313-22. · 9.82 Impact Factor
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2011, p. 3809–3818
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 77, No. 11
Hpa2 Required by HrpF To Translocate Xanthomonas oryzae
Transcriptional Activator-Like Effectors into Rice
Yu-Rong Li,1,2‡ Yi-Zhou Che,1‡ Hua-Song Zou,2‡ Yi-Ping Cui,1Wei Guo,1Li-Fang Zou,2
Eulandria M. Biddle,3Ching-Hong Yang,3* and Gong-You Chen1,2*
Department of Plant Pathology, Nanjing Agricultural University/Key Laboratory of Monitoring and Management for
Plant Diseases and Insects, Ministry of Agriculture of China, Nanjing 210095,1and School of Agriculture and
Biology, Shanghai Jiaotong University/Key Laboratory of Urban Agriculture (South), Ministry of Agriculture,
Shanghai 200240,2China, and Department of Biological Sciences, University of Wisconsin—Milwaukee,
Milwaukee, Wisconsin 532113
Received 6 December 2010/Accepted 23 March 2011
Xanthomonas oryzae pv. oryzicola, the causative agent of bacterial leaf streak, injects a plethora of effectors
through the type III secretion system (T3SS) into rice cells to cause disease. The T3SS, encoded by the hrp
genes, is essential for the pathogen to elicit the hypersensitive response (HR) in nonhost tobacco and for
pathogenicity in host rice. Whether or not a putative lytic transglycosylase, Hpa2, interacts with a translocon
protein, HrpF, to facilitate bacterial pathogenicity remains unknown. Here we demonstrated that both the hpa2
and hrpF genes are required for the pathogenicity of X. oryzae pv. oryzicola strain RS105 in rice but not for HR
induction in tobacco. The expression of hpa2 was positively regulated by HrpG and HrpD6 but not by HrpX.
In vivo secretion and subcellular localization analyses confirmed that Hpa2 secretion is dependent on HpaB (a
T3SS exit protein) and that Hpa2 binds to the host cell membrane. Protein-protein assays demonstrated that
Hpa2 interacts with HrpF. In planta translocation of AvrXa10 indicated that the mutation in hpa2 and hrpF
inhibits the injection of the HpaB-dependent transcriptional activator-like (TAL) effector into rice. These
findings suggest that Hpa2 and HrpF form a complex to translocate T3S effectors into plant cells for
pathogenesis in host rice.
Xanthomonas oryzae pv. oryzicola, the causative agent of
bacterial leaf streak disease in rice, is one of the model organ-
isms for studying the molecular mechanisms of plant-pathogen
pathosystems (41, 59). The bacterial ability to trigger the hy-
persensitive response (HR), a rapid and localized programmed
cell death in nonhosts or in resistant hosts, and to be patho-
genic in host plants depends on a type III secretion system
(T3SS) encoded by a 27-kb hrp cluster containing 10 hrp, 9 hrc
(hrp-conserved), and 8 hpa (hrp-associated) genes according to
the homologous regions in other Xanthomonas species (9, 41,
59). Some of the hrp-hrc-hpa gene products comprise a pedes-
tal-like T3SS structure that traverses the two bacterial mem-
branes (21, 24), a pilus-like secretion channel (HrpE) outside
HrcC (52), and a translocon protein (HrpF) in the eukaryotic
host membrane (3, 4, 6, 21, 48). As a whole, the T3SS appa-
ratus injects a number of effectors into the apoplast and cytosol
of eukaryotic host cells, including harpins, which elicit HR
induction in nonhost apoplasts (4, 45, 58), and transcriptional
activator-like (TAL) effectors, which lead to disease suscepti-
bility in hosts or trigger disease resistance in nonhosts upon
interaction with a specific R gene product surveillance system
(4, 11, 38, 39, 40, 49, 56). The virulence of Xanthomonas oryzae
pv. oryzae is markedly reduced when hrpF is mutated (48).
However, hrpF mutation has not been investigated in X. oryzae
The hpa genes contribute to virulence, but strains with mu-
tations in hpa genes generally do not exhibit phenotypic
changes in disease symptoms of the same severity as those with
other hrp-hrc gene mutations (9, 25, 29). Some Hpa proteins,
such as HpaB and HpaC from Xanthomonas campestris pv.
vesicatoria and T3SS exit proteins that promote the secretion
of a large set of effectors and prevent the delivery of nonef-
fectors into the plant cell, are indispensable for plant-pathogen
interactions (5, 19). In X. oryzae pv. oryzae, diverse phenotypes
displayed in rice by hpa gene mutants suggest that Hpa pro-
teins have distinct individual functions (9).
hpa2 is the first gene upstream of the core hrp cluster, be-
yond the hrpA operon of X. oryzae pv. oryzicola (59), and
similar gene arrangements are also found in the genomes of X.
oryzae pv. oryzae (57), X. campestris pv. vesicatoria (43), and
Xanthomonas axonopodis pv. glycines (29). Unfortunately, the
roles of the hpa2 homologs of these Xanthomonas species in
their proliferation in host tissues, virulence in host plants, and
HR induction in nonhosts are inconsistent with each other (7,
29, 43, 57, 58). The predicted Hpa2 protein, which belongs to
the lysozyme-like family of proteins, is proposed to dissolve or
disrupt the bacterial cell wall (31, 57) and is homologous to
* Corresponding author. Mailing address for Gong-You Chen:
School of Agriculture and Biology, Shanghai Jiaotong University, 800
Dongchuan Road, Shanghai 200240, China. Phone: 86-021-34205873.
Fax: 86-021-34205873. E-mail: email@example.com. Mailing address
for Ching-Hong Yang: Lapham Hall, Room S181, 3209 N. Maryland
Ave., Milwaukee, WI 53211. Phone: (414) 229-6331. Fax: (414) 229-3926.
‡ Y.-R.L., Y.-Z.C., and H.-S.Z. contributed equally to this work.
† Supplemental material for this article may be found at http://aem
?Published ahead of print on 8 April 2011.
VirB1 in Agrobacterium tumefaciens, which promotes the for-
mation of the type IV secretion system (T4SS) (39). However,
there is no evidence to demonstrate either that Hpa2 is se-
creted through the T3SS, where it localizes within the plant, or
with which proteins it interacts.
The expression of hrp genes is induced in planta and in the
hrp-inducing medium XOM3 (55) and is controlled by two key
regulatory genes, hrpG and hrpX, located outside the hrp gene
cluster (59). Commonly, the expression of the hrpA operon and
hrpX is activated by an OmpR family member, HrpG (42, 54).
HrpX, an AraC-type transcriptional activator, controls the ex-
pression of operons hrpB to hrpF, which are located down-
stream of genes encoding T3S effectors (53). Many HrpX regu-
lons possess a plant-inducible promoter (PIP) box 30 to 32 bp
upstream of a conserved ?10 box-like sequence (13, 14, 50).
Since there is an imperfect PIP box in the hpa2 promoter
region of X. oryzae pv. oryzicola (59), it is not clear whether
hpa2 is regulated by HrpX.
Having investigated the questions discussed above, we ad-
duce evidence that the loss of pathogenicity of an X. oryzae pv.
oryzicola hpa2 hrpF double mutant in susceptible rice is con-
sistent with a model in which a lack of Hpa2-HrpF complex
formation prevents the translocation of HpaB-dependent TAL
effectors into plants.
MATERIALS AND METHODS
Bacterial strains and growth conditions. The bacterial strains and plasmids
used in this study are listed in Table S1 in the supplemental material. The
wild-type X. oryzae pv. oryzicola strain RS105, X. oryzae pv. oryzae PXO99A, and
other Xanthomonas strains were grown on NA (0.5% peptone, 0.1% yeast, 1%
sucrose, 0.3% beef extract, and 1.5% agar) or NB (NA without agar) medium at
28°C. Escherichia coli and Agrobacterium tumefaciens strains were grown in
Luria-Bertani medium at 37°C and 28°C, respectively (36). The hrp-inducing
medium for X. oryzae strains is XOM3 (D-xylose,1.8 g/liter; D,L-methionine, 670
?M; sodium L-glutamate, 10 mM; NaFe2?-EDTA, 240 ?M; MgCl2, 5 mM;
KH2PO4, 14.7 mM; MnSO4, 40 ?M [pH 6.0]) (55). Yeast strains were grown in
YPD medium (Clontech), and the positive yeast clones for the yeast two-hybrid
(Y2H) system were screened on selective dextrose (SD) medium lacking ade-
nine, leucine, tryptophan, and histidine (SD/-Ade/-Leu/-Trp/-His) and on SD/
-Leu/-Trp/-His at 30°C. Onion tissue was cultured on 1/2 MS medium (0.22%
Murashige and Skoog basal medium, 0.05% morpholineethanesulfonic acid
[MES], and 1.5% agar) at 28°C in the dark. Antibiotics were used at the following
concentrations: ampicillin (Ap), 100 ?g/ml; kanamycin (Km), 50 ?g/ml; rifampin
(Rif), 50 ?g/ml; spectinomycin (Sp), 100 ?g/ml.
DNA manipulation and plasmid construction. DNA isolation, restriction en-
zyme digestion, subcloning, electrotransformation, PCR, Southern blotting, and
Western immunoblotting were performed according to standard procedures
(47). The PCR primers for the genes discussed in this report are listed in Table
S2 in the supplemental material. The PCR products were first cloned into the
pMD18-T vector (TaKaRa, Dalian, China) and were then verified by sequencing.
DNA sequences were analyzed with VECTOR NTI software (Invitrogen).
To construct an hpa2 promoter–?-glucuronidase (GUS) fusion construct, the
entire promoter region (bp ?1 to ?216) upstream of the hpa2 open reading
frame (ORF) was amplified from the genomic DNA of X. oryzae pv. oryzicola
RS105 with primers phpa2-F and phpa2-R (see Table S2 in the supplemental
material), and the product was then fused with the gusA gene (37), which was
amplified with primers gusA-F and gusA-R (see Table S2). The fusion was then
cloned into pUFR034 (10) at the EcoRI site, giving phpa2GUS (see Table S1).
To generate Hpa2 with a c-Myc tag, hpa2 with its native promoter (?1 to ?216
bp upstream) was amplified from the genomic DNA of X. oryzae pv. oryzicola
RS105 by PCR with the Hpa2-F/Hpa2Myc-R primer set (see Table S2 in the
supplemental material). The amplified PCR product was then cloned into
pUFR034 at the EcoRI and KpnI sites in frame with a c-Myc epitope-encoding
sequence, generating pHpa2-c-Myc (see Table S1).
To detect the interaction between the HrpF and Hpa2 proteins by the Y2H
system, the hpa2 open reading frame was amplified from the genomic DNA of
strain RS105 with the hpa2-F1/hpa2-R1 primer set (see Table S2 in the supple-
mental material) and was ligated into pGADT-7 and pGBDT-7 at the EcoRI and
BamHI sites, giving pAHpa2 and pBHpa2 (see Table S1), respectively. Similarly,
the hrpF gene was amplified with the hrpF-F1/hrpF-R1 primer set (see Table S2)
and was cloned into pGADT-7 and pGBDT-7 at the NdeI sites, giving pAHrpF
and pBHrpF (see Table S1), respectively.
To express the c-Myc-tagged HrpF protein, the hrpF open reading frame was
c-Myc tagged at the 3? terminus by PCR amplification from the genomic DNA of
strain RS105 with the HrpFMyc-F/HrpFMyc-R primer set (see Table S2 in the
supplemental material). The product was then cloned into the pET30a (?)
vector at the NdeI and EcoRV sites, giving pHrpF-c-Myc (see Table S1). To
express the glutathione S-transferase (GST)-tagged Hpa2 protein, the entire
hpa2 ORF without its native stop codon was PCR amplified from RS105
genomic DNA with the hpa2-F2/hpa2-R2 primer set (see Table S2) and was
then cloned into the pET41a (?) vector at BamHI and XhoI, giving pGST-
Hpa2 (see Table S1).
To construct the binary vector expressing the Hpa2-YN fusion, we first used
PCR with primers hpa2-F3 and hpa2-R3 (see Table S2 in the supplemental
material) to amplify the full-length Hpa2 coding sequence without the native
stop codon and then fused this sequence upstream of the N terminus of the
yellow fluorescent protein (YFP) sequence, which encodes the N-terminal por-
tion of YFP (amino acids [aa] 1 to 155), at the BamHI and KpnI sites, giving
pHpa2-YN (see Table S1). Similarly, to construct the binary vector expressing
the HrpF-YC fusion, the full-length HrpF coding sequence without the stop
codon was amplified with the hrpF-F2/hrpF-R2 primer set (see Table S2) and
was fused upstream of the C terminus of the YFP sequence, which encodes the
C-terminal portion of YFP (aa 156 to 239), at the XbaI and KpnI sites, giving
pHrpF-YC (see Table S1).
To express Hpa2 in onion epidermal cells, the entire hpa2 gene was amplified
with the hpa2-F4/hpa2-R4 primer set and was cloned into the pA-GFP vector
with XhoI and SpeI, giving pHpa2-GFP. All plasmid constructs were verified by
Mutagenesis of the hpa2 and hrpF genes. To generate nonpolar mutations in
the hpa2 and hrpF genes of X. oryzae pv. oryzicola strain RS105, upstream and
downstream flanking fragments of hpa2 were amplified from RS105 genomic
DNA with the hpa2I-F/hpa2I-R and hpa2II-F/hpa2II-R primer sets (see Table
S2 in the supplemental material), respectively, and were then cloned into
pMD18-T vectors (TaKaRa, Dalian, China). After the upstream and down-
stream fragments were digested by BamHI and XbaI and by XbaI and SalI,
respectively, the two fragments were cloned into the suicide vector pKMS1 (27)
at the BamHI and SalI sites, giving pK?hpa2 (see Table S1). Correspondingly,
the upstream and downstream flanking fragments of hrpF were PCR amplified
with the hrpFI-F/hrpFI-R and hrpFII-F/hrpFII-R primer sets (see Table S2),
respectively, and were then cloned into pKMS1 at the BamHI and SphI sites,
resulting in pK?hrpF (see Table S1). These constructs were introduced into X.
oryzae pv. oryzicola RS105, and then single mutants of hpa2 and hrpF were
isolated according to the procedure described by Jiang et al. in 2009 (27). Then
pK?hpa2 was transformed into the R?hrpF mutant for the construction of a
double mutant, R?hpa2?hrpF, by following the same procedure. The mutants
were verified by PCR amplification with the hpa2I-F/hpa2II-R and hrpFI-F/
hrpFII-R primer sets (see Table S2) and by Southern blotting with the probes of
the hpa2 and hrpF genes, respectively. By following the same procedure, the
single and double mutants of hpa2 and hrpF in strain PXO99A(22) of X. oryzae
pv. oryzae, the causal agent of rice bacterial blight, were generated and named
P?hpa2, P?hrpF, and P?hpa2?hrpF, respectively (see Table S1).
Pathogenicity and HR assays. Hypersensitive response and pathogenicity as-
says were performed as described previously (59). X. oryzae and its derivatives
were assessed for their abilities to cause disease symptoms and to multiply in
IR24 and IRBB10 rice (Oryza sativa subsp. indica) plants by inoculation of rice
seedlings (2 weeks old) by use of needleless syringes and of adult rice (2 months
old) by leaf needling for X. oryzae pv. oryzicola strains and by leaf clipping for X.
oryzae pv. oryzae strains with bacterial suspensions that were adjusted to a
concentration of 3 ? 108CFU/ml. IR24 is susceptible to RS105 and PXO99A.
IRBB10, containing the Xa10 gene, is resistant to X. oryzae pv. oryzae strains if
they carry a matching avrXa10 gene (28, 56). Xanthomonad strains were tested
for their abilities to elicit an HR on Nicotiana benthamiana by infiltration of plant
tissue using needleless syringes with strains adjusted to a concentration of 3 ?
108CFU/ml. Plant responses were scored 24 h postinoculation for the HR in
tobacco, 3 days postinoculation (dpi) for water-soaking symptoms in rice seed-
lings, and 14 dpi for lesion lengths. Leaves that became brown in the infiltrated
area instead of water soaked indicated HR in rice (56). All plants were grown in
growth chambers at 25°C with a 12-h photoperiod. Experiments were repeated at
least three times.
3810LI ET AL. APPL. ENVIRON. MICROBIOL.
Measurement of bacterial growth ability in rice. Cell suspensions of RS105
strains adjusted to a concentration of 3 ? 108CFU/ml were infiltrated into
recently expanded leaves of 2-week-old IR24 rice with needleless syringes at
three spots on each leaf. Three 0.8-cm-diameter leaf discs were harvested with a
cork borer from each area after infiltration. After sterilization in 70% ethanol
and 30% hypochlorite, the discs were ground with a sterile mortar and pestle into
1 ml of distilled water, diluted, and plated to determine the CFU/cm2. Serial
dilutions were spotted in triplicate onto NA plates with appropriate antibiotics.
Plates were incubated at 28°C for 3 to 4 days until single colonies could be
counted. The number of bacterial CFU per square centimeter of leaf area was
then estimated, and the standard deviation was calculated using colony counts
from the three triplicate spots from each of the three samples per time point per
inoculum. Experiments were repeated at least three times.
Promoter activity assays and reverse transcription-PCR (RT-PCR) analysis.
For GUS activity assays, X. oryzae pv. oryzicola strain RS105 and hrp mutants
were cultured in XOM3 to an optical density at 600 nm (OD600) of 0.5. Bacterial
cells were diluted and disrupted in sonic buffer (20 mM Tris-HCl [pH 7.0], 10
mM 2-mercaptoethanol, 5 mM EDTA, and 1% Triton X-100). GUS activities
were determined at 30-min intervals for 3 h by measuring absorbance at 415 nm
(A415) using p-nitrophenyl-D-glucuronide as the substrate (26). One unit was
defined as 1 nmol of 4-methyl-umbelliferone produced per min per bacterium.
For RT-PCR analysis, the bacteria were cultured as described for the GUS
activity assay. Total RNA was extracted by using the Trizol reagent according to
the manufacturer’s instructions (Invitrogen, Carlsbad, CA). Total RNAs were
quantified by measuring the OD260/OD280ratio and were analyzed by gel elec-
trophoresis. Before synthesis of the first strands, total RNAs were treated with
RNase-free DNase I (TaKaRa, Dalian, China) to remove potential traces of
genomic DNAs. To confirm the removal of contaminating DNA, extracted
RNAs were used as templates to amplify the target genes discussed in this report
with the primers listed in Table S2 in the supplemental material. cDNA synthesis
and PCR were conducted with AMV and Ex Taq DNA polymerases (both from
TaKaRa, Dalian, China) with primers hpa2-F and hpa2-R. PCR was performed
with a cycler using the following cycle parameters: 36 cycles of 94°C for 35 s, 52°C
for 35 s, and 72°C for 20 s. The resulting amplification products were analyzed in
1.2% agarose gels. The 16S rRNA gene of X. oryzae pv. oryzicola was used as the
internal control to verify the absence of significant variation at the cDNA level
in three samples.
Type III secretion assays. To detect the secretion of T3S effectors through the
type III system, X. oryzae pv. oryzicola strains were preincubated in NB medium
to logarithmic phase. Bacterial cells were harvested, adjusted to an OD600of 2.0
with sterilized water, and washed twice. Then 40 ?l of the bacterial suspension
was inoculated into 1 ml of modified XOM3 (55) and was incubated at 28°C for
16 h. Cell and supernatant fractions were separated by centrifugation, and the
protein in the supernatant fraction was precipitated with 12.5% trichloroacetic
acid (33). Proteins were separated on 10% sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) gels and were transferred to membranes
for immunoblotting using anti-c-Myc primary antibodies (Genescript, Nanjing,
China). Primary antibodies were recognized by anti-rabbit secondary antibodies
(Genescript, Nanjing, China) and were visualized on autoradiographs with the
Western-Light chemiluminescence system (Transgene, Beijing, China).
Y2H and ?-gal assays. Yeast two hybrid assays were conducted according to
the manufacturer’s instructions (Clontech, CA). Construct pairs consisting of
pAHpa2 and pBHrpF or of pBHpa2 and pAHrpF (see Table S1 in the supple-
mental material), either in a prey vector or in a bait vector, were transformed
into yeast strain AH109 in order to examine whether Hpa2 interacts with HrpF.
The positive clones on SD/-Ade/-Leu/-Trp/-His/ were confirmed by a ?-galacto-
sidase (?-gal) assay based on the manufacturer’s (Clontech, CA) manual (12).
GST pulldown assays. GST pulldown assays were performed as described
previously (5). pGST-Hpa2 and pHrpF-c-Myc were expressed in E. coli
BL21(DE3). Similarly, GST was expressed from the pET41a (?) vector (Nova-
gen, WI). Bacterial cells from 20 ml of cultures were resuspended in 2 ml
phosphate-buffered saline (PBS) and were broken by sonication. Insoluble cell
debris was removed by centrifugation, and soluble proteins were immobilized on
a glutathione resin according to the manufacturer’s instructions (Genescript,
Nanjing, China). A total of 30 ?l of GST lysate and 400 ?l of GST-Hpa2 lysate
were incubated with 50 ?l of resin according to protein stabilities and/or expres-
sion levels. Unbound E. coli proteins were removed by two washes with PBS.
After incubation with 600 ?l of the HrpF-c-Myc lysate for 1 h at 37°C, unbound
proteins were removed by centrifugation, and the resin was washed four times
with PBS containing 1% Triton X-100. HrpF-c-Myc was eluted with 30 ?l of 10
mM reduced glutathione at room temperature for 2 h. Three microliters of
total-protein lysates and 10 ?l of a solution of eluted proteins were analyzed by
SDS-PAGE and Western blotting using anti-c-Myc and anti-GST antibodies.
BiFC assays. For bimolecular fluorescence complementation (BiFC) assays,
plasmids were transferred into Agrobacterium tumefaciens GV3101 by a freeze-
thaw method (23). For infiltration, 50-ml cultures of Agrobacterium grown in LB
broth supplemented with 10 mM MES and 20 mM acetosyringone were har-
vested, washed, and resuspended in a solution containing 10 mM MgCl2, 10 mM
MES, and 100 mM acetosyringone. Tobacco (N. benthamiana) leaves from
3-month-old plants were used for infiltration. Five plants (two leaves per plant)
were infiltrated per experiment. After 12 to 16 h, Bisbenzimide H was infiltrated
into tobacco leaves. Four hours later, YFP fluorescence in tobacco leaves was
observed and imaged under a fluorescence microscope (Olympus IX71). The
excitation wavelength used for YFP was 488 nm, and the emission filter wave-
length was 520 to 550 nm. For visualization, tobacco leaf pieces were mounted
directly onto glass slides in a drop of water. For each experiment, at least 10
different samples were examined under the microscope. Experiments were re-
peated three times.
Subcellular localization of the Hpa2-GFP fusion protein. For subcellular lo-
calization, the full-length Hpa2 coding region was inserted into a pA-GFP vector,
which generated a C-terminal fusion with the green fluorescent protein (GFP)
gene under the control of the double cauliflower mosaic virus (CaMV) 35S
promoter. pA-GFP (control) and pHpa2-GFP were transiently expressed in
onion epidermal cells following transformation with a biolistic particle delivery
system (PDS-1000; Bio-Rad), using gold particles coated with column-purified
(Axygen) plasmid DNA, according to the manufacturer’s instructions. Onion
cells were cultured for 48 h on 1/2 MS medium and were subsequently bom-
barded under a slight vacuum using a helium pressure of 1,100 lb/in2. After
bombardment, plates were incubated at 28°C for 24 h. The subcellular localiza-
tion of the Hpa2-GFP fusion protein was observed using an MRC-1024 confocal
laser scanning microscope (Bio-Rad, CA).
Hpa2, together with HrpF, is essential for the pathogenicity
of X. oryzae pv. oryzicola in rice but not for the HR in tobacco.
The predicted Hpa2 protein in X. oryzae pv. oryzicola RS105
contains an N-terminal signal peptide of 34 amino acids and is
almost identical to Hpa2 from X. oryzae pv. oryzae (98% iden-
tity; 97% similarity) (59) and to HpaH (91% identity; 89%
similarity) from X. campestris pv. vesicatoria (43). These pro-
teins belong to the lytic transglycosylase family, which includes
VirB1 in A. tumefaciens, which promotes T4SS formation (39),
and HrpH and HopP1 in Pseudomonas syringae pv. tomato
DC3000; the latter suppresses pathogen-associated molecular
pattern (PAMP)-triggered immunity (PTI) in plants (44). It
has been shown that the formation of a T3SS and efficient
translocation of XopF1 are affected by the lysozyme-like HpaH
in X. campestris pv. vesicatoria (7), and X. oryzae pv. oryzae
Hpa2 has lytic activity against bacterial cell walls (57). These
reports prompted us to investigate whether Hpa2 may work
together with the translocator HrpF for bacterial pathogenicity
in rice. To determine the role of hpa2 in the pathogenesis of X.
oryzae pv. oryzicola in rice, we deleted hpa2, hrpF, or both the
hpa2 and hrpF genes in the wild-type strain RS105 to generate
the single mutants R?hpa2 and R?hrpF and the double mu-
tant R?hpa2?hrpF, respectively (see Table S1 in the supple-
To analyze HR induction, nonhost tobacco (N. benthami-
ana) leaves were inoculated by infiltrating bacterial suspen-
sions adjusted to a concentration of 3 ? 108CFU/ml. The
results of the HR assay showed that R?hpa2, R?hrpF, and
R?hpa2?hrpF elicited HR symptoms similar to those induced
by the wild-type strain, while a T3SS-deficient mutant, R?hrcV
(see Table S1 in the supplemental material), used as a control,
did not trigger the HR (Fig. 1A). This result demonstrated that
Hpa2 and HrpF are not involved in HR induction in nonhost
tobacco and that the secretion of the HR-inducing factor(s) is
VOL. 77, 2011X. ORYZAE pv. ORYZICOLA Hpa2-HrpF INTERACTION3811
affected in the hrcV mutant, but not in the single hpa2 and hrpF
mutants or in the hpa2 hrpF double mutant.
To investigate the roles of hpa2 and hrpF in the pathogenesis
of X. oryzae pv. oryzicola, we infiltrated the strains at a con-
centration of 3 ? 108CFU/ml into rice seedling leaves (Oryza
sativa cv. IR24, 2 weeks old) and monitored the plants for
water-soaked symptoms and for bacterial growth in planta.
Bacterial strains were also inoculated into adult rice plants (2
months old), and the lengths of the lesions formed were mea-
sured. The results revealed that the hrpF mutant, R?hrpF, still
caused weak water-soaked symptoms in rice seedlings, as did
the R?hpa2 mutant (Fig. 1B), but the R?hrpF bacterial leaf
streak lesions in adult plants were significantly shorter than
those caused by R?hpa2 (Fig. 1B). The growth of R?hrpF in
rice tissue was significantly reduced (P, 0.01 by t test) from that
of the hpa2 mutant (Fig. 1C). In contrast, the double mutant
R?hpa2?hrpF was unable to cause water-soaked symptoms in
rice seedlings (Fig. 1B) and was also deficient for patho-
genicity in adult rice, like R?hrcV. The in planta growth of
R?hpa2?hrpF was similar to that of R?hrcV in rice (Fig. 1C).
Complementation of R?hpa2?hrpF with either hpa2 or hrpF
restored water-soaked symptoms in rice seedlings, and patho-
genicity and bacterial growth in adult rice tissues, to the levels
caused by either R?hpa2 or R?hrpF, correspondingly (Fig. 1).
Overall, the results discussed above demonstrated that hpa2
together with hrpF is essential for the pathogenicity of X.
oryzae pv. oryzicola in rice, but not for HR induction in non-
host tobacco, suggesting that some T3S effectors contributing
to HR induction are not translocated into host cells.
Expression of hpa2 is positively regulated by HrpG and
HrpD6 but not by HrpX. hpa2 is the first gene left of the
pathogenicity island and beyond the hrpA operon of X. oryzae
FIG. 1. The association of hpa2 with hrpF is essential for X. oryzae pv. oryzicola RS105 to grow in rice and to cause disease on host plants.
(A) HR induced on nonhost plant tobacco (N. benthamiana) leaves by infiltration of plant tissue with strains adjusted to 3 ? 108CFU/ml by use
of a needleless syringe. Three replications were done in each experiment, and each experiment was repeated three times. (B) Water-soaking
symptoms caused by X. oryzae pv. oryzicola strains on inoculated leaves of the host plant, IR24 rice (a susceptible cultivar). Photographs were taken
3 days after inoculation into seedling rice (14 days old) by needleless syringe, and lesion length was measured 14 days after inoculation into adult
rice (2 months old) by the leaf-needling method (59). Bars with the same capital letter are not significantly different. (C) Growth of bacteria in
inoculated leaves. Bacteria were recovered from the inoculated leaves every day for a period of 4 days postinoculation. Data are means ? standard
deviations from three replicates.
3812LI ET AL.APPL. ENVIRON. MICROBIOL.
pv. oryzicola (59). Intriguingly, the expression profiles of the
hrp-hrc-hpa mutants (10 hrp, 9 hrc, and 8 hpa genes and 2 hrp
regulatory genes, hrpG and hrpX) of X. oryzae pv. oryzicola
revealed that hpa2 expression was greatly reduced in the hrpD6
mutant, R?hrpD6 (data to be published elsewhere). This
prompted us to investigate the hpa2 expression pattern in this
pathogen. The DNA sequence 31 bp upstream of the hpa2
locus shows that there is an imperfect PIP box (TTCGC-N15-
TTCGT) and also a ?10 box-like motif (TATGTT) (Fig. 2A).
HrpX is known to directly bind the PIP box so as to regulate
the transcription of hrp genes (30), and the presence of an
imperfect PIP box upstream of hpa2 implies that its expression
is positively regulated by HrpX. To determine whether the
expression of hpa2 with a predicted PIP box-regulated pro-
moter does indeed depend on HrpX, we performed an RT-
PCR analysis of X. oryzae pv. oryzicola RS105, the hrpG mu-
tant R?hrpG, the hrpX mutant R?hrpX, and the hrpD6 mutant
R?hrpD6 grown in the hrp-inducing medium XOM3, using
R?hpa2 as the negative control. As shown in Fig. 2B, the hpa2
transcript is highly abundant both in the wild-type strain RS105
and in R?hrpX but not in R?hrpG, R?hrpD6, or R?hpa2 (Fig.
2B). This indicates that hpa2 is not regulated by HrpX but
most likely by HrpG and HrpD6.
Additionally, the hpa2 promoter-driven ?-glucuronidase
(gusA) transcriptional fusion reporter plasmid phpa2GUS (con-
region fused to the promoterless gusA gene with its ribosome
binding site [RBS] in the pUFR034 vector) (see Table S1 in
the supplemental material) was introduced into R?hrpG,
R?hrpX, R?hrpD6, and RS105 by biparental conjugation using
Escherichia coli S17-1 as described previously (10, 30).
Transconjugants were screened on rich NA medium supple-
mented with appropriate antibiotics. The resulting reporter
strains were named R?hrpG(phpa2GUS), R?hrpX(phpa2GUS),
R?hrpD6(phpa2GUS), and RS105(phpa2GUS) (see Table S1).
In X. oryzae pv. oryzae, the expression of hrp genes, including the
regulators hrpG and hrpX, is induced in nutrient-deficient me-
dium but is repressed in nutrient-rich medium (13, 14, 15, 16).
Therefore, we measured the GUS activities of the reporter strains
NA medium. The results showed that the GUS activities of the
phpa2GUS reporter plasmid in the hrpG and hrpD6 mutant back-
grounds were significantly (P, 0.01 by t test) lower than that in the
hrpX mutant and in the wild-type background (Fig. 2C), indicat-
ing that hpa2 expression is induced, and its transcription is posi-
tively regulated, by HrpG and HrpD6, but not by HrpX, in X.
oryzae pv. oryzicola.
The secretion of Hpa2 is T3SS dependent. Hpa2, like HpaH
in X. campestris pv. vesicatoria (7, 43), shares sequence identity
with lytic transglycosylases. Information obtained from the se-
quence analysis prompted us to investigate whether or not
there are N-terminal amino acid patterns typical of T3S sub-
strates as described by Furutani et al. (14). In the first 50 amino
acid residues at the N terminus of Hpa2, there are a total of 8
Ser and Pro residues (less than 10), only 2 Leu residues, and no
Asp or Glu residues within the first 12 residues, and the fourth
residue is a Ser (data not shown), indicating that Hpa2 may be
secreted through the T3SS. To investigate the secretion char-
acteristics of Hpa2 in X. oryzae pv. oryzicola RS105, hpa2 was
expressed as a C-terminally c-Myc epitope tagged derivative in
plasmid pHpa2-c-Myc (see Table S1 in the supplemental ma-
terial) and was introduced into the wild-type strain RS105 and
a T3SS-deficient mutant, R?hrcV. Immunoblotting indicated
that a functional T3SS is necessary for the secretion of Hpa2,
since the Hpa2-c-Myc protein (expected size, 20 kDa for
Hpa2 plus 5 kDa for the c-Myc epitope) was not detected in
the culture supernatant (SN) of R?hrcV but was present in the
total-cell extract (TE) when bacteria were incubated in the
secretion medium XOM3 (Fig. 3A).
Since HpaB is an exit protein for the T3SS, we sought to
investigate whether the Hpa2 secretion via the T3SS is affected
by a mutation in hpaB. Thus, plasmid pHpa2-c-Myc was intro-
duced into the hpaB deletion mutant R?hpaB and the wild-
type strain RS105 (see Table S1 in the supplemental material).
After incubation in the secretion medium XOM3, the TEs and
SNs were analyzed by immunoblotting using an anti-c-Myc
antibody. Hpa2-c-Myc was detected only in the TE of the
R?hpaB culture, not in the SN, while it was detectable in both
FIG. 2. Expression of hpa2 in X. oryzae pv. oryzicola strain RS105 when the hrpG, hrpX, or hrpD6 gene is mutated. (A) Schematic map of the
promoter region containing the PIP box and ?10 box-like motif of hpa2 fused with GUS. (B) Detection of hpa2 expression by RT-PCR. RS105
and the hrpG, hrpX, and hrpD6 mutant strains were incubated in the hrp-inducing medium XOM3, and semiquantitative RT-PCR was performed.
PCR products were electrophoretically separated on a 1.2% agarose gel. The 16S rRNA PCR product was used as a control. (C) GUS activities
of the hpa2 promoter-GUS reporter in RS105, R?hrpG, R?hrpX, and R?hrpD6. Strains were cultured in either XOM3 or NB medium for 16 h,
and GUS activities were then determined by measurement of the OD415using p-nitrophenyl-?-D-glucuronide as a substrate. The experiment was
repeated twice, and similar results were obtained. Statistically different data groups are indicated by different letters.
VOL. 77, 2011X. ORYZAE pv. ORYZICOLA Hpa2-HrpF INTERACTION3813