全 文 ：Received 15 Aug. 2003 Accepted 18 Feb. 2004
Supported by the National Natural Science Foundation of China (30370969, 30230240), the Century-Across Excellent Talent Foundation
(Jiaokehan 2002, No. 48), and the State Key Basic Research and Development Plan of China (2003CB114204).
* Author for correspondence. Tel: +86 (0)25 84396072; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (9): 1083-1090
HarpinXoo and Its Functional Domains Activate Pathogen-inducible
Plant Promoters in Arabidopsis
PENG Jian-Ling, BAO Zhi-Long, LI Ping, CHEN Guang-Yong, WANG Jin-Sheng, DONG Han-Song*
(Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China)
Abstract: Harpins are bacterial proteins that can enhance plant growth and defense against pathogens
and insects. To elaborate whether harpins perform the diverse functions in coordination with the activa-
tion of specific promoters that contain particular elements, we cloned pathogen-inducible plant promoters
PPP1, PPP2, and PPP3 from tobacco and investigated their responses to harpinXoo or its truncated
fragments DEG, DIR, and DPR (domains for enhancing plant growth, insect resistance and pathogen resistance).
PPP1 contains an internal repeat composed of two tandem 111 bp fragments; 111 bp in the repeat was
deleted in PPP2. PPP3 contains a bacteria-inducible element; PPP1 and PPP2 additionally contain TAC-1
and Eli boxes inducible correspondingly by salicylic acid (SA) and elicitors. Function of cloned PPPs was
confirmed based on their activation in transgenic Arabidopsis plants by Ralstonia solanacearum (Ralston)
or SA. HarpinXoo, DEG, DIR, or DPR activated PPP1 and PPP2 but not PPP3, consistent with the presence
of Eli boxes in promoters. PPP1 was ca. 3-fold more active than PPP2, suggesting that the internal repeat
affects levels of the promoter activation.
Key words: harpinXoo; pathogen-inducible plant promoter (PPP); cis-acting elements; transgenic
Arabidopsis
Harpins produced by some plant pathogenic bacteria
can enhance plant growth and induce resistance to patho-
gens and insects by activating distinct signaling pathways
(Dong et al., 1999; Galán and Collmer, 1999; Kim and Beer,
2000; Peng et al., 2003) in many plants. HarpinXoo was iden-
tified in Xanthomonas oryzae pv. oryzae (Wen and Wang,
2001) as a harpin containing 140 amino acids with size of
15.6 kD. HarpinXoo affects plants similarly as other harpins
(Wen and Wang, 2001). Analysis of truncated harpinXoo
fragments has identified several functional domains, includ-
ing those for enhancing plant growth (DEG) and inducing
insect resistance (DIR) and pathogen resistance (DPR),
which contain 59, 33, and 49 amino acids, respectively (Li,
2002). Whether harpins and the functional domains per-
form the diverse functions by activating specific promot-
ers is unclear. In this context, activation of inducible plant
promoters is modulated by specific cis-acting elements. The
GCC Box AGCCGCC, W Box (T)TGAC(C/T), and S Box
AGCCACC are inducible by pathogens (Rushton and
Somssich, 1998; Rushton et al., 2002; Wang et al., 2002).
Distinctly, the TCA-1 Box (TATTCTT and ATCTTC) re-
sponds to SA, while Eli Box 1 (ATGGAT, ATATATG, or
TATCCA), Eli Box 3 (AACCGCAC and TTGGTTAA), and
Eli Box L (TCTAATCCAATC, ACTAACATCAAG or
TTAGGTTTC) are inducible by biotic elicitors (Gough et
al., 1995). Here we show that harpinXoo and the domains
activate pathogen-inducible plant promoters (PPPs) that
contain the Eli boxes.
1 Materials and Methods
1.1 Plants and bacteria
Nicotiana tabacum L. variety Xanthi was grown for 40
d before use (Dong and Beer, 2000). Arabidopsis thaliana
L. ecotype Columbia (Col-O) was grown in incubators with
a 14-h-day (200 mE.m-2.s-1 at 24 ℃) and 10-h-night (20
℃)cycle (Peng et al., 2003). Escherichia coli (Escherich
T) and Agrobacterium tumefaciens (Conn H J) were lyo-
phil ized and maintained at –80 ℃. Ralston ia
solanacearum was maintained in sterile water at room
temperature. Bacteria were multiplied as described
(Gerhardt et al., 1981).
1.2 Promoter cloning and analyses
BLAST searches for pathogen and hormone-inducible
promoters recognized regions conserved only in the pro-
moter ParAt of the auxin-inducible gene parAt (GenBank
accession number D42119) and the promoter MSR of the
multiple stimulation response gene str246C (X80829)
cloned from the tobacco variety Samsun. Accordingly, three
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041084
pairs of primers (5-GCAAGCTTATTGATGAAAATTTTG-
ACAT-3, 5-GTGGATCCAGTAGTACTATTTATAGTGAT-
ATATATTT-3; 5-GCAAGCTTTTGACCTTTTTCGATTCT-
3, 5-GTGGATCCAGTAGTACTATTTATAGTGATATAT-
ATTT-3; 5-GCAAGCTTGAAAAGGGTCCAGTCC-3, 5-
GTGGATCCAGTAGTACTATTTATAGTGATATATATTT-
3) were synthesized to clone PPPs that could cover differ-
ent regions of MSR. To facilitate cloning of PCR products
and construction of transformation units, the restriction
enzyme HindⅢ and BamHⅠ recognition bases were
added, respectively, to 5-termini of the upstream primers
and 3-termini of the downstream primers. Using high fidel-
ity polymerase Ex Taq (TaKaRa Biotech Co., Ltd., Dalian,
China), target sequences were amplified by PCR (Clark, 1997)
from genomic DNA of Xanthi. They were cloned into the
vector pET30 (a) and sequenced (TaKaRa Biotech Co., Ltd.)
or into pUC18 for further use, and both vectors were trans-
ferred into the E. coli strain DH5a by electroporation (Peng,
2003; Peng et al., 2003). Products of 1 308, 1 281 and 220 bp
from PCR with the first, second, and third pair of primers
were designated as PPP1 (GenBank accession number
AF469482), PPP2 (AF469481), and PPP3 (AF469483). The
standard BLAST and MegAlign (DANStar Inc., USA) pro-
grams were applied to compare and align sequences
(Fig.1A). The presence of cis-acting elements recognized
as per reports (Gough et al., 1995; Thomma et al., 1998) in
PPPs was searched by the Microsoft Word Search tool and
located by the BioEdit version 4.7.8 (Dept Microbiol, North
Carolina State University, USA).
1.3 Transgenic plant generation and promoter activation
assays
Each of PPPs in pUC18 was cloned into the vector
pBI121, which harbors the promoter 35S, the kanamycin-
resistance gene (NPTII), and the gus gene uidA (Jefferson
et al., 1987; Lazo et al., 1991), between the HindⅢ and
BamHⅠ sites to replace 35S (Fig.1B). After correct orienta-
tion was confirmed by sequencing, each of PPP::gus and
35S::gus was transferred by electroporation (Peng, 2003)
into the A. tumefaciens strain EHA105. Arabidopsis plants
were transformed by blossom infiltration (Clark, 1997; John
et al., 1997). For each transformation, 35 kanamycin-resis-
tant lines were selected by sequentially screening T1 and
T2 seeds on MS medium (Sambrook and Russell, 2001) sup-
plied with 50 mg/mL kanamycin. Integration of promoters
into the plant genome was determined by PCR.
Subsequently, homozygous T3 plants of 18 lines of each
PPP-transformed plant that resembled the wild type in mor-
phology were assayed for promoter activation, compared
to the wild type and the line 1 of 35S-transformed plants.
For plant treatment, inactive proteins used as a control,
harpinXoo, and the three fragments were produced, as de-
scribed (Li, 2002; Peng et al., 2003), from E. coli strains
harboring vectors pET30(a), pET30(a)::DEG, pET30(a)::DIR,
and pET30(a)::DPR, respectively. Plants were investigated
at 18 h, except specified elsewhere, after inoculation with R.
solanacearum by drenching roots with a bacterial suspen-
sion (107 cfu/mL) or spray with water or each of aquatic
solutions of 0.1 mmol/L SA, 15 µg/mL of harpinXoo, DEG,
DIR, DPR, and the control. GUS staining in leaves was ob-
served by microscopy (Belbahri et al., 2001) and the level
of promoter activity quantified by fluorescent spectropho-
tometry (Clark, 1997). Experiments were done 3–5 times each
involving 5 plants and subjected to analysis by Microsoft
Excel T test (P ≤0.05).
2 Results
2.1 Sequence analyses of PPPs
BLAST searches revealed a 95%-98% identity of PPPs
with matched regions in ParAt and MSR but no identity
with other promoters from plants. PPP1 and PPP2 match the
region spanning the sites 771 and 2 075 while PPP3 matches
1 084-2 075 nucleotides in MSR, which has a full-length of
2 207 nucleotides (Gough et al., 1995). Figure 1A shows
comparison of the promoters and recognition of cis-acting
elements in PPPs. First, PPP1 contains an internal repeat
composed of two tandem 111 bp fragments, while 111 bp in
the repeat was absent in PPP2. PPP2 also lacks 20 nucle-
otides at the 3-teminus of the first repeat. Second, align-
ments disclosed more differences between PPPs. PPP2 and
PPP3 are short of four nucleotides (GGAT) that appear at
the 3-terminus of PPP1. PPP2 also lacks a thymine and two
adenine bases resided at the sites 390, 1 284 and 1 294 in
PPP1, while two adenine bases present in PPP1 (at sites 1
275 and 1 276) and PPP2 (at sites 1 144 and 1 145) are absent
in PPP3. Finally, a bacteria-inducible element (Bac) is lo-
cated downstream of PPPs and MSR. PPP1 and PPP2 also
contain a pair of W Box, which resides in PPP3 as a single
motif, a pair of TCA-1 motif, an Eli Box 1, an Eli Box 3, and a
pair of Eli Box L. Thus, PPPs differ from MSR and one an-
other in components of nucleotides and elements.
2.2 Confirmation of PPP functions
Though Gough et al. (1995) elaborately assayed func-
tions of several elements in MSR, whether PPPs are active
requires to be determined, because they were isolated from
a different variety of tobacco, cover partial sequences of
MSR, and were different from each other. We transformed
Arabidopsis plants with each of PPPs (Fig.1B, C) and in-
vestigated transgenic plants for inducibility of PPPs by R.
PENG Jian-Ling et al.: HarpinXoo and Its Functional Domains Activate Pathogen-inducible Plant Promoters in Arabidopsis 1085
solanacearum and SA. First, based on replicate assays,
the three PPPs are activated by the bacterial infection. In
PPP-transgenic plants, GUS staining was not observed with-
out inoculation (Fig.2A-C), while staining was evident in
leaves following inoculation of roots (Fig.2D-G). As a posi-
tive control, the 35S promoter was markedly active in plants
with and without inoculation (Fig.2G, H). By contrary,
staining was not evident in wild-type plants (Fig.2I). Ac-
tivities of PPP1, PPP2, and PPP3 were ~53, ~39, and ~25
times that of 35S, while PPP1 and PPP2 were 3- and 2.5-fold
more active than PPP3, in inoculated plants (Fig.3A). These
results suggest that the Bac element plays a role in modu-
lating response of PPPs to bacterial infection. Moreover,
PPP functions were also confirmed by determining their
Fig.1. Pathogen-inducible plant promoter (PPP) analyses and engineering performance. A. Comparison of PPPs with the promoter
MSR. PPP1 is aligned with full length PPP2, PPP3 and partial MSR region spanning the nucleotide site 771 and the 3-terminus. The 111
bp sequence repeated tandem up and down stream are indicated by solid and open triangles, respectively. Nucleotides involved in the
labeled elements are top-lined. The Bac is bordered with arrows. B. Transformation constructs. C. PCR analysis of PPPs present in
transgenic plants. Gels show products of PPP1 (top), PPP2, and PPP3 (bottom). Sizes in bp of markers (M) are on the left. Over the top,
WT refers to the wild type and numbers are codes of transgenic lines.
Acta Botanica Sinica 植物学报 Vol.46 No.9 20041086
responses to SA (Fig.3B). In wild-type plants, background
readings of 800-900 arbitrary units remained unchanged
over time after treatment. Similarly, GUS activity was not
greater than background readings in PPP3 plants, consis-
tent with the absence of the TCA-1 motif in the promoter.
Inversely, activities of PPP1 and PPP2 increased markedly
in 24 h post-SA application, being 4.3- and 2-fold greater
than that of 35S. Thus, TCA-1 seems to bestow SA respon-
siveness on the promoters.
2.3 Activation of PPPs by harpinXoo and its functional
domains
We addressed whether harpinXoo and its functional do-
mains can activate PPPs that contain Eli boxes, by compar-
ing GUS activities in plants following spray with the pro-
teins (Fig.4). Background read similarly in different geno-
types treated differently. However, GUS activities were sig-
nificant in PPP1 or PPP2 plants but not evident in PPP3
plants, as compared with the wild type. Moreover, we found
that PPPs responded to DEG, DIR, and DPR similarly as did
to harpinXoo. Both PPP1 and PPP2 were significantly acti-
vated by DEG, DPR, or DIR. By contrary, PPP3 did not
display any response to the three fragments. These results
suggest that harpinXoo and the domains activate PPPs in
consistence with the putative role of Eli boxes.
Fig.2. Visualization of PPP activation in leaves of transgenic
plants. GUS staining was observed in PPP (A-F) and 35S-trans-
formed plants (G, H), compared with the wild type (I). Codes of
transgenic lines tag with dashes after names of promoters. NT
and Rs mean non-treatment and inoculation with Ralstonia
solanacearum.
Fig.3. Levels of pathogen-inducible plant promoter (PPP) acti-
vation by R. solanacearum and SA. PPP activities in leaves were
quantified by fluorescent spectrophotometry and compared to the
constitutive 35S activity. Background readings were monitored in
wild-type plants treated similarly. A. Assays done at 24 h after
inoculation with the bacterium. B. Assays for treatment with SA.
Fig.4. Levels of pathogen-inducible plant promoter (PPP) acti-
vation by the intact and truncated harpinXoo. Plants were sprayed
with indicated proteins and assayed for GUS activities in leaves
24 h later. DEG, DIR and DPR, domains for enchancing plant
growth, insect resistance and pathogen resistance.
PENG Jian-Ling et al.: HarpinXoo and Its Functional Domains Activate Pathogen-inducible Plant Promoters in Arabidopsis 1087
2.4 Levels of PPP activities as affected by the internal
repeats
To indicate whether the internal repeats affect the pro-
moter activation, we compared levels of PPP1 and PPP2
activities induced by R. solanacearum, SA, harpinXoo or
each of the three domains. PPP1 was better inducible than
PPP2 by every factor. Relatively, PPP1 was ~1.4- and ~2-
fold more active while induced by bacteria (Fig.3A) and SA
(Fig.3B), and ~1.9-, ~1.5-, and ~1.3-fold more active while
induced by harpinXoo, DEG, and DPR (Fig.4), respectively.
Thus, the deletion of the internal repeat decreases the acti-
vation of the promoter.
3 Discussion
We cloned three PPPs and studied their activation in
transgenic Arabidopsis plants with two results. First, PPPs
contain a Bac element and are activated by R.
solanacearum. Second, PPP1 and PPP2 also contain SA
and elicitor-inducible elements, while PPP3 does not.
Consistently, SA, harpinXoo, and the three functional do-
mains of harpinXoo can activate PPP1 and PPP2, but they
fail to activate PPP3.
Our results indicate roles of several cis-acting elements
in modulating the promoter activation. First, the Bac ele-
ment present in PPPs and MSR (Gough et al., 1995) is
identical, while ParAt region matched with the Bac sequence
lacks five consensus motifs (Niwa et al., 1994). Whereas
studies here and reported previously (Gough et al., 1995)
have demonstrated the systemic activation of PPPs and
MSR by R. solanacearum, which infects Arabidopsis and
other plants from roots (Gerhardt et al., 1981; Belbahri
et al., 2001), whether ParAt can respond to pathogens is
unclear (Menke et al., 1999). In the context, our results
could not indicate whether the W Box plays a role in the
activation of PPPs. Second, TCA-1 boxes seem to impart
SA inducibilities to PPPs, because SA activates PPP1 and
PPP2 but does not activate PPP3, consistent with the in-
duction of MSR by SA due to TCA-1 boxes (Gough et al.,
1995). Finally, Eli boxes seem to be required for the activa-
tion of PPPs by harpinXoo, because PPP1 and PPP2 are ac-
tive but PPP3 is not in response to the protein. In addition,
PPP1 with two tandem 111 bp fragments is more active than
PPP2 that lacks an 111 bp region, suggesting that the inter-
nal repeat can strengthen the function of PPP1. However,
the elements in PPPs show consensus obviously distinct
from GCC-like boxes and the S Box, which are inducible
correspondingly by oomycetes and fungi or fungal elici-
tors (Menke et al., 1999; Eulgem et al., 2000; Ohme-Tgaki et
al., 2000). The consensus in elements of PPPs also is differ-
ent from the JERE and DRE Box TACCGAC, which are me-
diated by jasmonates and environmental stresses (Datta
and Muthukrishnan 1999; Menke et al., 1999). How these
distinct elements coordinate promoter responses to differ-
ent inducers remains to be studied.
The activation of PPP1 and PPP2 but not PPP3 by
harpinXoo domains indicates a possible linkage between
roles of Eli boxes and the domains. Based on elaborate
studies (Li, 2002), DEG is 2.2-fold better than harpinXoo in
enhancing plant growth but ~30% and ~5% reduced in in-
ducing resistance to insects and pathogens. DPR is 2.3-
fold more effective than harpinXoo for plant growth and
pathogen resistance but is ~20% impaired for insect
resistance. DIR is similar to harpinXoo in inducing insect
resistance and ~50% compromised in enhancing plant
growth. Thus, in response to harpinXoo or the domains,
insect resistance seems less coordinately with pathogen
resistance while more coordinately with plant growth. This
notion is consistent with the dogma that distinct signaling
pathways lead to resistance to pathogens and insects
(Clarke et al., 1998; 2000; Orozco-Cárdenas et al., 2001;
Rairdan et al., 2001). However, harpinXoo and the domains
activate PPP1 and PPP2 by a similar extent. Possibly, the
domains act similarly as does an intact harpinXoo regarding
recognition by Eli boxes. Alternatively, Eli boxes may be
unable to recognize differences between the domains due
to lacks of definitive consensus in the domains (Galán and
Collmer, 1999; Kim and Beer, 2000), given that complemen-
tary motifs usually are required for interactions between
nucleic acids and protein partners (Dangl and Jones, 2001).
Cloning of bacteria-inducible plant promoters has been
in paucity, relative to those inducible by other pathogens
(Martini et al., 1993; Pontier et al., 1994; Strittmatter et al.,
1996; Manners et al., 1998; Coutos-Thevenot et al., 2001;
Ayliffe et al., 2002). PPP3 is the shortest bacteria-inducible
promoter cloned from plants thus far. The activation of
PPPs by harpinXoo could allow for many strategies to engi-
neer crops for the improvement of multiple traits.
Acknowledgements: We thank Dr. Beer S V (Department
of Plant Pathology, Cornell University, Ithaca), and Dr. LIU
De-Hu (The Chinese Academy of Agricultural Sciences,
Beijing) for gifts of the R. solanacearum strain, and the
EHA105 strain and pBI121 vector. We appreciate critical
comments on this article by two anonymous reviewers.
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(Managing editor: ZHAO Li-Hui)