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: Gongyou Chen[Show abstract] [Hide abstract]
ABSTRACT: Xanthomonas oryzae pv. oryzicola (Xoc) is the causal agent of bacterial leaf streak, a devastating disease in rice. Xoc uses a type III secretion (T3S) system, which is encoded by the hrp-hrc-hpa (hypersensitive response and pathogenicity, hrp-conserved and hrp-associated) genes, to inject repertoires of T3S effectors (T3Es) into plant cells. Many of the hrp-hrc-hpa genes have roles in pathogenesis, but the role of hrpE3, which shows homology to hpaE in X. campestris pv. vesicatoria (Xcv), is poorly understood. In this study, hrpE3 was shown to be transcribed independent of the hrpD operon, and its expression was dependent on a promoter within hpaB. The expression of hrpE3 was positively regulated by HrpG and HrpX, a finding probably caused by an imperfect plant-inducible promoter (PIP) box (TTCGT-N16 -TTCGA) in the hrpE3 promoter. The secretion of HrpE3 was dependent on T3S, and subcellular localization of HrpE3 was cytoplasmic and nuclear in plant cells. A mutation in hrpE3 reduced the virulence of Xoc by decreasing disease lesion length and bacterial growth in planta. Full virulence was restored to the mutant when Xoc hrpE3, but not Xcv hpaE, was expressed in trans. The differences in transcription, secretion via the T3S system and bacterial virulence in plants were attributed to N-terminal amino acid differences between Xoc HrpE3 and Xcv HpaE. Collectively, the results demonstrate that hrpE3 encodes a T3E protein which is delivered into the plant cell through the T3S system, localizes to the cytoplasm and nucleus, and is required for full virulence in rice.Molecular Plant Pathology 05/2013; · 3.88 Impact Factor
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ABSTRACT: The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9≤X≤25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism.PLoS ONE 01/2014; 9(3):e93205. · 3.73 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The type III secretion system (T3SS), encoded by hrp (hypersensitive response and pathogenicity) genes in Gram-negative phytopathogenic bacteria, delivers repertoires of T3SS effectors (T3SEs) into plant cells to trigger the hypersensitive response (HR) in nonhost or resistant host plants and promote pathogenicity in susceptible plants. The expression of hrp genes in Xanthomonas is regulated by two key regulatory proteins, HrpG and HrpX. However, the interactions between hrp gene products in directing T3SE secretion are largely unknown. Here we demonstrated that HrcT of X. oryzae pv. oryzicola (Xoc) functions as a T3SS component and positively regulates the expression of hrpX. Transcription of hrcT occurs via two distinct promoters, one is with the hrpB operon (T1) and the second (T3) within hrpB7. Via either T1 or T3 promoter, the defect in Hrp phenotype by hrcT deletion was restored in the presence of hrcT only from Xanthomonas species, but not other phytopathogenic bacteria. An N-terminally truncated HrcT was able to bind the hrpX promoter and activate the expression of hrpX, supporting that HrcT is a positive regulator of hrpX. A revised model showing the regulatory interactions between HrcT, HrpX and HrpG is proposed.Applied and environmental microbiology 04/2014; · 3.69 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: firstname.lastname@example.org. 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.
3810 LI 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 INTERACTION 3813
the TE and the SN of strain RS105 (Fig. 3A), suggesting that
Hpa2 secretion is HpaB dependent.
The fact that hpa2 expression is positively regulated by
HrpG and HrpD6, but not by HrpX, prompted us to investi-
gate whether hpa2 is secreted in the hrpG, hrpX, and hrpD6
mutants. The Hpa2-c-Myc fusion protein was expressed in
R?hrpG, R?hrpX, and R?hrpD6 (see Table S1 in the supple-
mental material). After incubation in an hrp-inducing medium
for 16 h, a protein of the expected size was detected by a c-Myc
epitope-specific antibody in the TEs and SNs of the bacterial
strains tested. The results showed that the Hpa2-c-Myc fusion
protein was detected in the SNs of the wild-type strain RS105
and the R?hrpX mutant only, not in R?hrpG, R?hrcV, and
R?hrpD6 (Fig. 3A). Comparing these results with the hpa2
expression profiles of the hrpG, hrpX, and hrpD6 mutants (Fig.
2), we conclude that Hpa2 is a member of the HrpD6 and
HrpG regulon secreted through the T3SS but is not a member
of the HrpX regulon.
Hpa2 interacts with HrpF in the plasma membranes of
plant cells. In X. campestris pv. vesicatoria, the injection of T3S
effectors into plant cells is mediated by a translocon protein,
HrpF, which is the last extracellular component of the T3SS
appendage to penetrate the membrane of plant cells (6, 7, 46).
The fact that the hpa2 and hrpF double mutant R?hpa2?hrpF
lost its pathogenicity in rice (Fig. 1) suggests that HrpF re-
quires Hpa2 to form a complex for the translocation of T3S
effectors into plant cells. To test this hypothesis, we utilized the
yeast two-hybrid (Y2H) system to determine whether Hpa2
interacts with HrpF. Our Y2H assay showed that Hpa2 inter-
acts with HrpF (Fig. 4A), and this was true no matter which
protein (Hpa2 or HrpF) was used as the bait (data not shown).
To confirm the interaction, we expressed a pGST-Hpa2 chi-
mera in E. coli, immobilized it on glutathione Sepharose, and
added an E. coli lysate containing pHrpF-c-Myc (Fig. 4B). The
result showed that HrpF-c-Myc specifically bound to GST-
Hpa2 but not to GST alone (Fig. 4B), which strongly suggested
that Hpa2 and HrpF interact with each other. To evaluate
whether Hpa2 and HrpF self-interact, we also tested whether
Hpa2-c-Myc specifically bound GST-Hpa2 and whether HrpF-
c-Myc coeluted with GST-HrpF. Indeed, no such self-interac-
tions occurred (data not shown).
Since X. campestris pv. vesicatoria HrpF is a translocator
that binds to the membranes of host cells (6), the interaction
between Hpa2 and HrpF prompted us to determine the cellu-
lar localization of Hpa2 once it is secreted via the T3SS. GFP-
tagged Hpa2 was transiently expressed in onion epidermal
cells, and the localization was examined by fluorescence mi-
croscopy. Hpa2 exhibited a plasma membrane localization that
is distinguishable from that of GFP (Fig. 5A). Interestingly, we
observed unique punctate staining of Hpa2 in the membranes
of onion cells before or after plasmolysis, and Hpa2-GFP did
not colocalize with the cell wall in the plasmolyzed cell (Fig.
5A), suggesting that Hpa2 binds the membranes of plant cells.
To further confirm that the interaction of Hpa2 with HrpF
occurs in the plasma membrane of the host, we performed a
bimolecular fluorescence complementation (BiFC) technique
(51) to visualize Hpa2-HrpF association in plant cells. For
BiFC, the nonfluorescent N-terminal domain of YFP (YN)
and the nonfluorescent C-terminal domain of YFP (YC) were
fused to the N- and C-terminal ends of the test proteins (i.e.,
Hpa2 and HrpF), respectively. Interactions between the re-
spective fusion proteins were then tested in N. benthamiana
using the Agrobacterium-mediated transient expression assay.
Representative BiFC confocal microscopy images showing the
FIG. 3. Detection of the secretion of Hpa2 and the hpaB-dependent T3S effector AvrXa10 in X. oryzae pv. oryzicola by immunoblotting.
(A) Strains RS105, R?hrcV, R?hrpG, R?hrpX, R?hrpD6, and R?hpaB expressing pHpa2-c-Myc were incubated in secretion medium. Total-
protein extracts (TE) and culture supernatants (SN) were analyzed by immunoblotting using anti-c-Myc antibodies. (B) Strains RS105, R?hrcV,
R?hpa2, R?hrpF, R?hpa2?hrpF, and R?hpaB harboring AvrXa10-FLAG were incubated in secretion medium. TEs and SNs were analyzed by
immunoblotting using anti-FLAG antibodies.
FIG. 4. Protein-protein assays to detect interaction of Hpa2 with
HrpF of X. oryzae pv. oryzicola. (A) Interactions of Hpa2 and HrpF
were evaluated using full-length proteins by Y2H and ?-gal assays. The
reactions of pGADT7-T with pGBKT7-53 and of pGADT7-T with
pGBKT-Lam were regarded as the positive (?) and negative (?)
controls, respectively. Y2H and ?-gal assays were performed according
to standard procedures (Clontech). For the image shown, Hpa2 was
used as the bait. Similar results were seen when HrpF was used as the
bait. (B) Hpa2 interacts with HrpF by a pulldown assay. GST and
GST-Hpa2 were immobilized on glutathione Sepharose and were in-
cubated with HrpF-c-Myc. Total-cell lysates (TE) and eluted proteins
(Eluate) were analyzed by immunoblotting using antibodies directed
against the c-Myc epitope and GST, respectively. Bands corresponding
to GST and GST chimeras are marked by asterisks. Arrows indicate
c-Myc epitope-tagged proteins.
3814LI ET AL.APPL. ENVIRON. MICROBIOL.
Hpa2-HrpF interaction are presented in Fig. 5B. Coexpression
of pHpa2-YN and pHrpF-YC resulted in bright fluorescence
near the plant cell membrane (Fig. 5B). Taken together, the
BiFC studies show that Hpa2 interacts with HrpF and that
localization occurs in the plant cell membrane.
Hpa2 and HrpF are involved in the translocation of TAL
effectors into host cells. It has been demonstrated that X.
oryzae pv. oryzae AvrXa10 is a TAL effector that triggers the
HR in a T3SS-dependent manner (48) on Oryza sativa cv.
IRBB10 (containing the corresponding R gene Xa10), and this
avr-R gene-mediated response is inhibited by T3S effectors of
X. oryzae pv. oryzicola (35). To investigate the roles of Hpa2
and HrpF in HR inhibition, we employed the hrp system of X.
oryzae pv. oryzae as the reporter. The avrXa10 gene was intro-
duced into the hpa2 and hrpF single mutants of X. oryzae pv.
oryzae strain PXO99A(P?hpa2 and P?hrpF), the hpa2 and
hrpF double mutant (P?hpa2?hrpF), and the T3S mutant
P?hrcV (see Table S1 in the supplemental material). Three
days after rice seedlings (2 weeks old) were syringe-infiltrated
with Xanthomonas strains at 3 ? 108CFU/ml, the innate
AvrXa10 in the wild-type X. oryzae pv. oryzae strain PXO99A,
P?hpa2, or P?hrpF could trigger a typical HR (e.g., the infil-
trated area became brown) in IRBB10 rice, which contains the
Xa10 R gene, but did not elicit the HR response when ex-
pressed in the double mutant P?hpa2?hrpF or in the T3SS
mutant P?hrcV (Fig. 6A). Furthermore, expression of N-ter-
minally truncated AvrXa10 (AvrXa1029-1201) or full-length
AvrXa10 in wild-type X. oryzae pv. oryzicola RS105 did not
stimulate the HR in IRBB10 rice (Fig. 6A). To confirm these
results, the strains listed above, except for the transconjugants
of wild-type RS105 with either the empty vector pURF034 or
plasmid pAvrXa10, were inoculated at 3 ? 108CFU/ml into
adult rice leaves (IRBB10) by the leaf-clipping method. Four-
teen days after inoculation, we observed that the avrXa10 gene
had transformed the wild-type PXO99A, P?hpa2, and P?hrpF
strains from compatibility to incompatibility with IRBB10 rice,
with lesion lengths less than 1.5 cm (Fig. 6B and C), but
N-terminally truncated AvrXa10 did not alter the compatibility
of the wild-type strain PXO99Awith IRBB10 rice. In addition,
no lesions were formed in IRBB10 rice by the double mutant
P?hpa2?hrpF or the T3SS mutant P?hrcV, even though they
harbored the innate avrXa10 gene (Fig. 6B and C). In contrast,
the avrXa10 gene did not alter the virulence of the wild-type
strain PXO99A, P?hpa2, or P?hrpF in IR24 rice, lacking the
matching Xa10 gene (Fig. 6C). These data indicate that a
single mutation in either hpa2 or hrpF does not inhibit the
translocation of AvrXa10 into plant cells but that a mutant
lacking both hpa2 and hrpF is unable to translocate AvrXa10,
which implies that both HrpF and Hpa2 contribute to the
translocation of T3S effectors into plant cells.
To verify that the secretion of the TAL effector AvrXa10 via
the T3SS is not affected by the absence of Hpa2 and HrpF,
AvrXa10 was expressed as a C-terminally FLAG epitope
tagged derivative in plasmid pAvrXa10 (see Table S1 in the
supplemental material), and this plasmid was introduced into
strains R?hpa2, R?hrpF, R?hpa2?hrpF, and RS105. When
the bacteria were incubated in XOM3 secretion medium,
AvrXa10-FLAG was detected by a FLAG epitope-specific an-
tibody in both the TEs and the SNs. The results showed that
AvrXa10-FLAG was detectable in the TEs of all the strains
tested (Fig. 3B), indicating that the protein is expressed. How-
ever, the fusion protein was undetectable only in the SNs of
R?hrcV and R?hpaB but was detectable in R?hpa2, R?hrpF,
and R?hpa2?hrpF (Fig. 3B), indicating that the AvrXa10 TAL
effector is an HpaB-dependent T3SS protein and that neither
Hpa2 nor HrpF plays a role in the secretion of AvrXa10 via the
T3SS but that both are involved in the translocation of the
effector into plant cells, as demonstrated above.
In this study, we showed that Hpa2 works as a helper for
HrpF to form a translocon complex on the plant membrane in
FIG. 5. Fluorescence assays to detect the localization of Hpa2 with
HrpF in plant cells. (A) Subcellular localization of Hpa2-GFP in onion
epidermal cells. Particle bombardment was used to transfect onion
cells with the pA-GFP (control) or Hpa2-GFP vector. Expression was
driven by the CaMV 35S promoter. For confocal laser scanning mi-
croscopy, samples were taken 24 h postinoculation. The plasmolysis of
cells expressing Hpa2-GFP was implemented in 30% (wt/vol) sucrose
solution for 15 min. Representative fluorescent images of cells express-
ing pHpa2-GFP are shown. Cytosolic GFP bombardment was per-
formed as the control. (B) BiFC visualization of Hpa2 and HrpF
association in a stably transformed N. benthamiana cell membrane. A.
tumefaciens GV3101 harboring p1301-YN or pHpa2-YN coinfiltrated
with p1301-YC or pHrpF-YC, respectively. Representative images
from microscopy show reconstituted YFP fluorescence. Bars, 10 ?m.
LM, light microscopy; FM, fluorescence microscopy.
VOL. 77, 2011 X. ORYZAE pv. ORYZICOLA Hpa2-HrpF INTERACTION 3815
order to facilitate the translocation of X. oryzae T3S effectors
into plant cells.
Hpa2 shares sequence identity with the lytic transglycosylase
family members Hpa2 in X. oryzae pv. oryzae (57), HpaH in X.
campestris pv. vesicatoria (7), HrpH, HopP1, and HopAJ1 in P.
syringae pv. tomato DC3000 (44), and VirB1 in A. tumefaciens
(60). The members of this predicted lytic transglycosylase fam-
ily do not contribute significantly to bacterial pathogenicity in
Shigella flexneri (1), P. syringae pv. tomato DC3000 (44), or X.
oryzae pv. oryzae (57) but enhance virulence in X. campestris
pv. vesicatoria and X. axonopodis pv. glycines (29, 43). The
efficient T3SS secretion and translocation of AvrBs3 in X.
campestris pv. vesicatoria depends not only on the global T3S
chaperone HpaB but also on HpaH (5, 7). This is consistent
with our hypothesis that the translocation of TAL effectors
through HrpF requires the participation of Hpa2. Possibly,
after secretion through the Hrp pilus, the lytic activity of Hpa2
may also dissolve the membranes of plant cells as it disrupts
plant cell walls (57) to aid in the insertion of HrpF into the
plant plasma membrane, which facilitates the transportation of
T3S effectors into plant cells. This hypothesis is based on our
subcellular localization analysis with GFP-labeled Hpa2, which
demonstrated that Hpa2 binds to the plasma membrane with
unique punctate staining (Fig. 5A), suggesting that HrpF to-
gether with Hpa2 forms a translocon complex in the mem-
branes of plant cells.
PIP box and ?10 box-like motifs are characteristic features
of HrpX regulons (14, 30, 50). Although there is an imperfect
PIP box and a ?10 box-like motif in the hpa2 promoter region
of X. oryzae pv. oryzicola RS105, the expression level of hpa2 in
the hrpX mutant is equivalent to that in wild-type RS105, but
hpa2 expression is significantly repressed in the hrpG mutant.
This demonstrates that hpa2 is regulated by HrpG rather than
by HrpX or that hpa2 is not naturally a member of the HrpX
regulon in X. oryzae pv. oryzicola. In addition, base substitu-
tions in the PIP box consensus sequences (TTCGC sequences)
have revealed that one or two base substitutions can consider-
ably suppress promoter activities. For example, when the last C
in the TTCGC sequence is replaced by T, promoter activity is
reduced by 50% (50). Contrasting results might be obtained
when base substitutions are made in PIP box sequences of
different genes in various bacteria. Remarkably, hpa2 is regu-
lated by HrpD6 in X. oryzae pv. oryzicola, suggesting that
additional regulatory pathways exist among these hrp genes.
The mutation of hpa2 in X. oryzae pv. oryzae (57) and of
hpaH in X. axonopodis pv. glycines (5, 7) resulted in an inability
to trigger HR in plants, in contrast to the mutation of hpa2 in
X. oryzae pv. oryzicola RS105. Otherwise, the X. oryzae pv.
oryzicola RS105 hpa2 mutant displayed reduced pathogenicity
in host rice similar to that of the hpa2 mutant of X. oryzae pv.
oryzae and the hpaH mutant of X. axonopodis pv. glycines (29,
57). Collectively, these data indicated that homologous genes
from different bacteria may play dissimilar roles during the
The HrpF protein of X. campestris pv. vesicatoria is consid-
ered a T3SS translocon protein, which inserts a translocation
FIG. 6. The translocation of effector protein AvrXa10 was obstructed by Hpa2 and HrpF. (A) The X. oryzae pv. oryzae-rice pathosystem was used
to check AvrXa10 secretion and translocation, since the AvrXa10-triggered HR is inhibited in X. oryzae pv. oryzicola (35). Strains at 3 ? 108CFU/ml
containing the fusion gene in pAvrXa10 were infiltrated into seedling leaves of IRBB10 rice (2 weeks old) with needleless syringes. The response in rice
was photographed 3 days after inoculation in three independent experiments. (B) Strains containing pAvrXa10 were inoculated into IRBB10 rice (2
months old) at 3 ? 108CFU/ml by leaf clipping. Photographs were taken 14 days postinoculation. (C) Lesion lengths in IRBB10 and IR24 rice, scored
14 days postinoculation. Data are means ? standard deviations from three repeats, each using 10 leaves. Bars with the same capital letter are not
significantly different. Lane or image numbers are as follows: 1, X. oryzae pv. oryzae PXO99A(pUFR034); 2, PXO99A(p?28AvrXa10); 3,
PXO99A(pAvrXa10); 4, P?hrcV(pAvrXa10); 5, P?hpa2(pAvrXa10); 6, P?hrpF(pAvrXa10); 7, P?hpa2?hrpF(pAvrXa10); 8, RS105(pUFR034); 9,
3816LI ET AL.APPL. ENVIRON. MICROBIOL.
channel into the eukaryotic plasma membrane (6). The patho-
genicity of the X. oryzae pv. oryzicola hrpF mutant showed
minimal virulence, distinguishing itself from other hrp-hrc mu-
tants, in accordance with the reduced virulence of the hrpF
mutant in X. oryzae pv. oryzae (9, 48), but not in X. axonopodis
pv. glycines (29). Thus, it could be expected that the reduced
pathogenicity of the hrpF mutant is due to incomplete channel
formation, which may occur in other components of the
translocon. Interestingly, the hpa2 hrpF double mutant of
RS105 completely lost pathogenicity in host rice but did not
prevent Hpa1, HrpB2 (data not shown), and the T3S effector
AvrXa10 from being secreted by the T3SS. Moreover, protein-
protein assays that revealed the interaction of Hpa2 with HrpF
strongly support our hypothesis that Hpa2 and HrpF form a
complex on the plant cell membrane to control the transloca-
tion of T3S effectors into the plant cell cytosol for bacterial
pathogenicity in hosts. However, further experiments are still
needed to clarify the complex mechanisms that control the
translocation of T3S effectors into plant cells.
Intriguingly, we also found that the hpa2 hrpF double mu-
tant retains the ability to trigger the HR in tobacco (Fig. 1A),
implying that the HR elicitor(s) of X. oryzae pv. oryzicola is not
translocated through the HrpF-Hpa2 complex into plant cells.
This is consistent with the finding that some T3SS substrates
are only secreted into the plant apoplast but not injected into
plant cells (35). The following evidence supports our hypoth-
esis. (i) Harpin proteins are apoplastic HR elicitors in plants,
since HR induction in tobacco occurs when the purified har-
pins are infiltrated into tobacco leaves (59). (ii) Gram-negative
plant-pathogenic bacteria have more than one HR elicitor for
HR induction in nonhost plants. For example, in P. syringae pv.
tomato DC3000, at least four proteins, HrpZ (20), HrpW (8),
HopAK1 (32), and HopP-1 (32), were able to induce the HR
in tobacco. In Ralstonia solanacearum, three HR elicitors,
PopA (2), HrpW (17), and PopW (34), have been reported.
(iii) Only the hpa1 gene product has been identified as a harpin
in X. oryzae pv. oryzicola (59). However, the hpa1 mutation in
X. oryzae pv. oryzicola does not render the pathogen unable to
trigger the HR in nonhost tobacco (unpublished data), in con-
trast to that in X. oryzae pv. oryzae (9), suggesting that there
are more than two harpins in Xanthomonas species. (iv) Hpa1
is secreted via the T3SS in an HpaB-independent manner (our
unpublished data) similar to that of its homolog XopA in X.
campestris pv. vesicatoria (43), but TAL effectors are HpaB
dependent, which explains why the hpa2 hrpF double mutant of
X. oryzae pv. oryzicola still triggers the HR in nonhost tobacco
but does not cause bacterial leaf streak in rice. The informa-
tion obtained from the mutations generated in hpa1, hpa2,
hpaB, and hrpF will serve as clues for the identification of an
additional HR elicitor(s) in X. oryzae pv. oryzicola.
We are grateful to Alan Collmer at Cornell University for critical
suggestions and helpful discussions on the experiments in this study.
This work was supported by the State Key Basic Research and
Development Project of China, the Natural Science Foundation of
China (30710103902 and 31071656), and the Special Fund for Agro-
Scientific Research in the Public Interest (NYHYZX07-056 and
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