Molecular Cloning and Functional Analysis of a Novel
Type of Bowman-Birk Inhibitor Gene Family in Rice1
Li-Jia Qu2, Jun Chen2, Meihua Liu, Naisui Pan, Haruko Okamoto3, Zhongzhuan Lin, Chengyun Li,
Donghui Li, Jinling Wang, Guofeng Zhu, Xin Zhao, Xi Chen, Hongya Gu, and Zhangliang Chen*
Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, National Laboratory of
Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing
100871, China (L.-J.Q., J.C., M.L., N.P., H.O., D.L., J.W., G.Z., X.Z., X.C., H.G., Z.C.); China Agriculture
University, Beijing 100094, China (Z.C.); Institute of Crop Breeding and Cultivation, Chinese Academy of
Agricultural Sciences, Beijing 100094, China (Z.L.); and Institute of Agro-Biotechnology, Yunnan Provincial
Academy of Agricultural Sciences, Kunming 650223, China (C.L.)
Bowman-Birk inhibitor (BBI) genes encode serine protease inhibitors that have repetitive cysteine-rich domains with reactive
sites for the trypsin or chymotrypsin family. We have identified seven BBI genes from japonica rice (Oryza sativa subsp.
japonica var Teqing). All of the genes identified were found in a single cluster on the southern end of the long arm of rice
chromosome 1. Four of the seven BBI genes have two repetitive cysteine-rich domains, whereas one has a truncated domain
with only one reactive site. We have also identified three novel BBI genes, each of which possesses three repetitive domains
instead of two. In situ hybridization analyses indicated that the accumulation of rice BBI transcripts was differentially
regulated in germinating embryos and also in the leaves, roots, and flower organs at later developmental stages. Different
members of the rice BBI gene family displayed different expression patterns during rice seed germination, and wounding
induced the expression of rice BBI transcripts. The three-domain BBIs had higher expression levels than the two-domain
BBIs. It was also found that the mRNA of rice BBI genes was present in abundant amounts in scutellar epithelium and
aleurone layer cells. RBBI3-1, one of the three-domain RBBI, exhibited in vitro trypsin-inhibiting activity but no
chymotrypsin-inhibiting activity. Overexpression of RBBI2-3 in transgenic rice plants resulted in resistance to the fungal
pathogen Pyricularia oryzae, indicating that proteinase inhibitors confer resistance against the fungal pathogen in vivo and
that they might play a role in the defense system of the rice plant.
Plants have developed defense systems to combat
various pathogens throughout their life cycle, from
the seed stage until senescence, and it is particularly
important that the embryo be kept free from infection. There are several embryonic defense mechanisms, including the production of plant lectins and
pathogen-related proteins in response to attacks by
pathogens or insects (Swegle et al., 1992; Ye et al.,
2001; Guiderdoni et al., 2002). A well-known defense
component is Ser protease inhibitors. They are expressed in developing seeds and are thought to play
an important role in inhibiting trypsin and chymotrypsin of external origin (Ryan, 1981). Two major Ser
protease inhibitors have been studied extensively in
1
This work was supported by the National High-Tech Program
of China (project no. GN 863– 01–101– 02– 02 and 2001AA222261 to
L.-J.Q.), by the National Natural Science Foundation (project no.
GN 39830020 to H.G.), by the State Key Basic Research and Development Plan (project no. GN G1999011602), and by the Rockefeller Foundation (grant no. GN#97003 to Z.L.C.).
2
These authors contributed equally to the paper.
3
Present address: Department of Plant Sciences, University of
Oxford, Oxford OX1 3RB, UK
* Corresponding author; e-mail [email protected]; fax 86 –10 –
6275–1841.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.103.024810.
560
plants: Kunitz inhibitors and Bowman-Birk inhibitors
(BBIs; Ryan, 1990). BBIs are Cys-rich proteins of
about 8 to 16 kD with disulfide bonds and are encoded by a family of related genes. The BBI gene
family has been found in both the Fabaceae and the
Poaceae. BBIs identified in Fabaceae, such as soybean
(Glycine max) and lima bean (Phaseolus lunatus), are
8-kD proteins. They have one BBI domain with two
reactive sites for trypsin and the related enzymes,
such as chymotrypsin (Birk, 1987). These protease
inhibitors are double-headed, with two reactive sites
in a single inhibitor molecule. Interestingly, this type
of inhibitor displays anticarcinogenic activity (Birk,
1993; Kennedy, 1993).
The BBIs found in barley (Hordeum vulgare) and
foxtail millet (Setaria italica) are either 8- or 16-kD
proteins (Nagasue et al., 1988; Tashiro et al., 1990).
The three-dimensional structures of the 8- and 16-kD
BBIs have been explored (Li de la Sierra et al., 1999;
Song et al., 1999). The 8-kD inhibitors found in these
monocotyledonous plants are single-headed, with
only one reactive site in the BBI domain, and inhibit
only trypsin. In contrast, the 16-kD inhibitors have
two homologous BBI domains with one reactive site
in each domain. It is therefore thought that the 16-kD
inhibitors evolved from the monocotyledonous 8-kD
single-headed inhibitors (Prakash et al., 1996).
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Rice Bowman-Birk Inhibitor Gene Family
The first amino acid sequence of a mature 16-kD
BBI protein from rice (Oryza sativa) was reported 15
years ago for the rice bran trypsin inhibitor (RBTI;
Tashiro et al., 1987). The cDNA of RBTI was later
identified (Chen et al., 1997) and characterized
(OsBBPI; Rakwal et al., 2001). Similar to other 16-kD
BBIs of monocotyledonous plants, RBTI also has two
homologous domains.
We have previously described the biochemical purification of RAFP1, an antifungal protein that is
produced by germinating rice seeds and displays
inhibitory activity on trypsin (Liu et al., 1994). The
N-terminal amino acid sequence of RAFP1 is homologous to the rice BBI, RBTI (Tashiro et al., 1987). In
this study, we have synthesized degenerate primers
and cloned a family of rice BBI genes. In addition to
the previously known types of BBIs, we have identified a novel type of BBI that has a third Cys-rich
domain in the N terminus with a potential trypsinreactive site. Significantly, the genes we have cloned
are clustered on the southern end of chromosome I,
although they possess different expression patterns.
Prokaryotic expression of RBBI3-1 shows that the
fusion protein has trypsin-inhibiting activity but no
chymotrypsin-inhibiting activity. Transgenic overexpression of the gene RBBI2-3 in rice cv Taipei 309
resulted in strong resistance in the seedling period to
the fungal pathogen responsible for rice fungus blast
disease, Pyricularia oryzae. The importance of the Ser
protease inhibitors in disease resistance will also be
discussed.
RESULTS
Cloning of Rice BBI Genes
Two degenerate primers were designed: The 5⬘
primer was designed based on the N-terminal amino
acid sequence of RAFP1 (Liu et al., 1994), whereas the
3⬘ primer was made according to the sequence of the
mature protein of a rice BBI, RBTI (Tashiro et al.,
1987). We performed a PCR using rice genomic DNA
as the template, with a 450-bp fragment being cloned
and sequenced. A clone designated as RBBI8 contained an open reading frame (ORF), and the deduced amino acid sequence was about 74% identical
to that of the rice BBI, RBTI (Xie et al., 1996). The
RBBI8 gene was expressed in Escherichia coli, and the
purified recombinant protein showed strong inhibitory activity against trypsin, as well as slight inhibitory activity against chymotrypsin (Li et al., 1999).
Using a recombinant F2 population of japonica rice
(Oryza sativa subsp. japonica cvs Moroberekan and
CO39), RBBI8 was found to cosegregate with a single
molecular marker RG612 that is located on the distal
southern end of rice chromosome 1 (Fig. 1A).
Southern-blot hybridization using the RBBI8 gene as
a probe showed a multiple band pattern in all three
restriction enzymes used in this analysis (Fig. 1B).
These results indicate that RBBIs in rice consist of a
multigene family of at least four members.
To clone all of the members of this gene family, a
rice bacterial artificial chromosome (BAC) library
constructed from the japonica rice var Teqing was
screened using RBBI8 as a probe. The four positive
BAC clones were found to be the same with respect
to patterns of restriction fragments. Fragments from
the BAC clone that hybridized with RBBI8 probes
were subcloned and sequenced (GenBank accession
nos. AJ277468, AJ277469, AJ277470, and AJ277472).
Within the BAC clone, a total of seven ORFs were
found to encode proteins that are homologous to
BBIs from both dicots and monocotyledon plants.
Denomination, Genomic Organization, and Sequence
Analysis of the Rice BBI Gene Family
The seven members of the rice BBI gene family
were found clustered in a BAC clone (33H2). All of
the BBI genes encoded Cys-rich domains. Similar to
previously identified BBI proteins, rice BBIs also contain homologous Cys-rich domains. Significantly,
these domains are repeated within a protein in most
rice BBIs. We have classified our RBBI clones based
on the number of the Cys-rich domain repeats (Fig.
1C). As shown in the Figure 1C, those with two
repeats were named RBBI2 and those of three repeats
were named as RBBI3.
The organization of rice BBI genes in the BAC clone
33H2 is shown in Figure 1D. All of the seven BBI
ORFs were tandem clustered on the same strand. The
two-domain class members RBBI2-1, RBBI2-2,
RBBI2-3, and RBBI2-4 encode proteins of 184, 193,
185, and 190 amino acid residues, respectively (Fig.
1E). The Cys-rich domain composition of the RBBIs
of this class has domain 1 with putative double reactive sites at its C terminus and domain 2 with one
putative reactive site. The three-domain class members RBBI3-1, RBBI3-2, and RBBI3-3 encode proteins
of 251, 259, and 254 amino acid residues, respectively
(Fig. 1E). The RBBIs of this class have an additional
homologous Cys-rich domain 3 with one putative
reactive site in the N terminus.
The RBBI8 gene is identical to the coding region of
RBBI2-3, which shows high similarity (from 72.5% to
85.3% identity) to other six members of RBBI gene
family at the DNA level. RBTI cDNA (Chen et al.,
1997; Rakwal et al., 2001) is identical to the RBBI3-3
gene in the 3⬘-flanking region (208 bp) and only
possesses a 6-bp difference in the coding region (data
not shown). The sequence differences between the
RBTI cDNA and RBBI3-3 are therefore most likely to
represent differences between orthologs of different
rice sub-species. Moreover, our sequence data
showed that the reported RBTI cDNA sequence
(Chen et al., 1997; Rakwal et al., 2001) could be incomplete because it is shorter than the RBBI3-3 transcript in the 5⬘-terminal region as determined by
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Qu et al.
Figure 1. Molecular cloning of the rice BBI gene family. A, Diagram of rice chromosome 1. The RBBI gene cluster is located
on the southern end of this chromosome as indicated by the arrow near the genetic marker, RG612. B, Southern blot using
RBBI8 (see “Materials and Methods”) insert (the conserved coding region of RBBI2-3, 450 bp in length) as a probe. Rice
genomic DNA was digested with BamHI (1), XhoI (2), BamHI ⫹ XhoI (3), and BamHI ⫹ EcoRV (4). C, Diagram showing the
structural composition of the RBBI family. D, Composition of the RBBI gene cluster on chromosome 1. The arrowhead
indicates the directions of the ORFs. Bar ⫽ 1 kb. E, Alignment of the conserved Cys-rich domains. Gaps are indicated by
a dash. Putative trypsin- and/or chymotrypsin-reactive sites are indicated in blue letters, and the conserved Cys residues are
in red.
reverse transcriptase (RT)-PCR and sequencing (M.
Liu, J. Chen, and L.-J. Qu, unpublished data).
Three more RBBI genes were further identified
from the genomic database of rice after draft sequences of rice genome were released (Goff et al.,
2002; Yu et al., 2002). Two of them have one domain,
and the other has two domains. The two singledomain RBBI genes were similar to Wip1, a woundinducible BBI gene found in maize (Zea mays), wheat
(Triticum aestivum), barley, and sorghum (Sorghum
bicolor; Tiffin and Gaut, 2001), and therefore were
designated as Oswip1-1 and Oswip1-2. The one with
two domains was similar to the RBBI2s we identified
and was therefore designated as RBBI2-0. Interestingly, these 10 RBBI genes were located in a locus of
rice chromosome 1 that spanned a region of about
430 kb (Fig. 1D).
Each RBBI interestingly has a highly hydrophobic
N-terminal sequence that shows weak homology
with the others (Fig. 1E) and that may serve as a
signal peptide. The N-terminal sequence for the twodomain members ranges from 52 to 55 amino acid
residues in length, but varies between 42 to 45 amino
acid residues for the three-domain members
(Fig. 1E).
Transcripts of Rice BBIs Can Be Detected in Many
Different Tissues
We used in situ hybridization utilizing the coding
region of RBBI2-3 as a probe to detect the overall
expression patterns of all of the rice BBI gene mem-
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Rice Bowman-Birk Inhibitor Gene Family
bers in different rice tissues. The data showed that
RBBI mRNA could be detected by the antisense
probe in many different tissues at different developmental stages (Fig. 2), whereas no signal was detected with the sense probe (data not shown). It was
found that mRNA started to accumulate in the embryo (both in the plumule and radicle) 24 h after
rehydration. The accumulation was mainly focused
in the coleoptiles, the first and the second leaves, and
the area around the growing point of the embryo bud
with a few transcripts from the mesocotyl to the
radicle (Fig. 2A). We detected differential expression
of RBBI mRNA in embryonic leaves. Although RBBI
genes are expressed in all leaf tissues in the embryo,
younger leaves had greater expression compared
with older leaves (Fig. 2B). After germination, RBBI
transcripts were detected mainly in mesophyll cells
within the leaves, with a much more reduced signal
in the vascular bundle compared with the embryonic
leaves (Fig. 2C). In the radicle, RBBI hybridization
signals were concentrated in the meristematic cells
close to the radicle cap (Fig. 2D). However, the mature root stem has less expression relative to the root
cap, and accumulation was mainly detected in the
endodermis and the vascular bundle (Fig. 2E). The
expression of the RBBI genes was also observed in
different flower organs such as the ovule, style, anther, petals, and pollen sac, and in pollen (Fig. 2, F–I).
Differential Expression Patterns within the Rice BBI
Gene Family
Northern blotting was carried out to study the gene
expression patterns of different gene members in
different rice organs using the 3⬘-untranslated region
(UTR) of each gene as a probe (Fig. 3). Expression of
two of the two-domain class genes, RBBI2-1 and
RBBI2-4, was not detected in any of the organs we
analyzed. RBBI2-2 was expressed slightly in leaves,
but was not detected in other organs. RBBI2-3 was
detected in roots and leaves, and in immature rice
grains, but was not detected in the stem or in flowers.
The three-domain class genes were more abundantly
expressed than the two-domain class genes. In particular, RBBI3-1 was highly expressed in roots (Fig. 3).
RBBIs Are Induced during Germination
Because the RAFP1 protein was first isolated from
germinating seeds, we analyzed the expression pattern of the RBBI genes during germination. RBBI
mRNA accumulation could be detected 24 h after
imbibition and reached a maximum at 48 h (Fig. 4A).
We also analyzed RBBI expression in germinating
seeds using in situ hybridization with the probe recognizing all of the RBBI genes. After rehydration of
the seed, RBBI transcripts were detected at a high
level in the scutellar epithelium that comprises the
Figure 2. Analysis of RBBI gene expression in rice tissues by in situ hybridization. Expression of RBBI genes was analyzed
by in situ hybridization using the conserved coding region of RBBI2-3 (450 bp) as a probe. A, Longitudinal section of an
embryo 24 h after rehydration, showing the embryo bud (EB) and radicle (R); B, transverse section of a young leaf at the base
of a 6-d-old seedling; C, transverse section of upper part of an old leaf (11 d old); D, radicle tip 24 h after rehydration,
showing the radicle cap (RC); E, transverse section of a primary root stem (from 11-d-old plants); F, close-up of the anther
at prophase of meiosis, showing pollen mother cells (PMC); G, transverse section of a flower during meiosis, showing the
pistil (Pi) and stamens (S); H, transverse section of pollen sacs (PS) at the anaphase of meiosis; and I, close-up of a pollen
sac after meiosis showing the tapetum (T) and immature pollen grains (P).
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Qu et al.
activity than that of the control, commercial soybean
BBI (Fig. 5A). However, no inhibitory activity against
chymotrypsin was observed (Fig. 5B).
Expression of BBI Genes Is Wound Inducible
The coding region of RBBI2-3 was used to study the
inducible expression of RBBI genes in rice leaves by
northern blotting. Because the probe sequence is a
conserved region showing 72.5% to 85.3% sequence
identity to other six members of RBBI family at the
DNA level, the detected signal is actually a mixed
signal. The experimental results show that the accumulation of some RBBI transcripts (especially the
two-domain transcripts) is wound inducible (Fig. 6).
This is consistent with a report that a rice BBI gene
OsBBPI (i.e. RBBI3-3) responded to cut, jasmonic
acid, and ethylene signals (Rakwal et al., 2001). The
probe used in that report consisted of a 259-bp conserved coding sequence and a 238-bp 3⬘ non-coding
region of RBBI3-3, and showed 96% and 83% identity
to the same regions of RBBI3-1 and RBBI2-3, respectively. Moreover, the 259-bp coding sequence of the
probe was 74%, 78%, 81%, and 84% identical to the
comparable coding regions of RBBI2-2, RBBI2-1,
RBBI3-2, and RBBI2-4, respectively. Therefore the detected wound-induced signals in that report could
also be a mixture, at least of RBBI2-3, RBBI3-1, and
RBBI3-3 (Rakwal et al., 2001). These results suggest
that RBBIs may be involved in the defense response
against pathogens and/or insects.
Figure 3. Differential expression patterns of RBBI genes. Northernblot analysis of RBBI genes. Gene-specific 3⬘-UTR probes of RBBI2-1,
RBBI2-2, RBBI2-3, RBBI2-4, RBBI3-1, RBBI3-2, RBBI3-3, and an 18S
ribosomal RNA probe were used to detect the gene expression in the
root (R), stem (S), leaf (L), flower (Fl), and young rice caryopsis (Fr).
layer of cells between embryo and endosperm (Fig.
4B). In addition, RBBI mRNA was abundant in aleurone cells (Fig. 4C), but only in the region relatively
close to the embryo and not in the entire aleurone cell
layer (Fig. 4D). The significance of this interesting
expression pattern needs to be further elucidated.
One of the Three-Domain BBIs, RBBI3-1,
Inhibits Trypsin
Knowledge of BBIs with three domains was hitherto unknown. The DNA fragment coding for the
three-domain of RBBI3-1 was amplified, cloned, and
expressed in E. coli to test whether the new threedomain RBBI is active as an inhibitor. The His-Tag
fusion protein, designated as F31, was purified before trypsin and chymotrypsin inhibitory assays
were performed. F31 showed strong inhibitory activity against trypsin and displayed stronger inhibitory
Overexpression of RBBI2-3 in Rice cv Taipei 309 Leads to
Resistance to the Rice Blast Fungus Pathogen
We have transformed the rice cv Taipei 309 with
RBBI2-3 to test the role of RBBI genes in plant defense
mechanisms. The rice cv Taipei 309 was chosen as a
host for this experiment because this cultivar is
known to be susceptible to various pathogens. We
transformed embryonic callus of rice cv Taipei 309
with RBBI2-3 by particle bombardment, and two
transgenic lines, T137-2 and T191-2, were obtained.
Integration of the transgene into the host rice genome
was confirmed by PCR and Southern blot (data not
shown). Two independent groups performed the resistance experiments simultaneously. The Beijing
group tested two isolates of P. oryzae, Zhong 10-8-14
and Sichuan 26, which are common in southern
China and severely infect rice cv Taipei 309. The
Kunming group tested four isolates of P. oryzae (i.e.
96-4-1a, 96-4-2a, 96-8-1a, and 96-10-1a) that are common in southwest China. For each resistance test, 100
seeds of each transgenic line, together with the nontransgenic rice cv Taipei 309 as a control, were
planted first on Murashige and Skoog medium and
then transferred to soil. Rice blast fungus pathogen P.
oryzae was inoculated onto rice leaves. Two days
after inoculation, semiquantitative RT-PCR was per-
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Rice Bowman-Birk Inhibitor Gene Family
Figure 4. Expression pattern of RBBI genes during germination. A, Northern-blot analysis of
RBBI genes. Accumulation of the RBBI mRNAs
was detected 24, 48, and 72 h after the onset of
seed imbibition. In situ hybridization results
showing RBBI gene expression in scutellar epithelium (SE; B), aleurone layer (AL; C), and
young embryo (EM; D) of imbibed germinating
rice seeds. The conserved coding region of the
RBBI2-3 gene (450 bp) was used as the probe.
formed. Six to 7 d after inoculation, the resistance of
the plants against the pathogens was evaluated.
Although 2,000 kilometers apart, the two independent groups obtained very similar results (Table I).
The semiquantitative RT-PCR analysis showed a
higher expression of RBBI2-3 in transgenic rice leaves
relative to controls (Fig. 7A). As expected, analysis of
pathogen effects showed that untransformed rice
plants of rice cv Taipei 309 were severely infected,
showing many typical spindle-shaped lesion spots
on the leaves and a severely retarded growth (Fig.
7B). However, the transgenic T137-2 and T191-2 grew
healthy and normally after the inoculation of fungal
pathogen. Not even a single small lesion spot was
found on all of the leaves of the transgenic seedlings,
indicating that the transgenic lines were highly resistant (resistance level 0; Mackill and Bonman, 1992)
to all isolates of the blast fungus pathogen tested
(Fig. 7B).
DISCUSSION
A genomic clone containing seven full-length rice
BBI genes is reported in this study. The genomic
organization of the RBBI gene family became clear
after three more RBBI genes were found in the databases of draft rice genome sequences (Fig. 1D).
Among the different RBBI members, the most obvi-
ous difference is the number of domains a gene contained. We report here for the first time that rice BBIs
contain one, two, or three BBI domains, although
there are reports that monocot (such as wheat) BBIs
possess either one or two domains (Odani et al.,
1986). The standard mechanism, exhibited by inhibitors when they react with cognate proteinases, indicates that the P1 residue in the reactive loop determines specificity of the inhibitor (Laskowski and
Kato, 1980). Several different P1 residues were detected in the RBBI domains. Those Wip1-like RBBIs,
single-domain inhibitors, have Phe as the N-terminal
P1 residue, indicating that these inhibitors tend to
inhibit chymotrypsin-like enzymes. The N-terminal
P1 residue for the multidomain RBBIs is Arg or Lys
in most cases and shows inhibiting activities against
trypsin-like enzymes (Fig. 1E) as determined inhibitory activity assays (Fig. 5). However, some P1 residues are changed from alkaline amino acid residues
to others. For example, the P1 residue of the first
domain of RBBI3-3 is changed to Met, which theoretically may change its inhibitory activity to target
chymotrypsin-like enzymes. In another case, the P1
of the second domain of RBBI2-4 is changed to Ser,
making the inhibitor more likely to inhibit elastaselike enzymes (Fig. 1E). More varieties are found in
other residues of the reactive loops, indicating that
the effectiveness of those proteinase-inhibiting doFigure 5. Titration assay of trypsin- and
chymotrypsin-inhibiting activity of F31 and soybean BBI. A, One hundred twenty-two TAME
units of trypsin was incubated with increasing
amounts of inhibitors in 0.2 mL of 0.05 M TrisHCl buffer, pH 8.0, at 25°C for 10 min. Trypsin
activity was then determined. The reaction volume was 1 mL. B, Chymotrypsin (2.0 ␮g) was
incubated with increasing amounts of inhibitors
in 0.2 mL of 0.05 M Tris-HCl buffer, pH 7.5, at
25°C for 10 min. Chymotrypsin activity was then
determined. The reaction volume was 1 mL.
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Figure 6. RBBI genes are wound inducible. The conserved region of
RBBI2-3 (350 bp) was used as a probe to detect transcripts of all of
the RBBI2 and RBBI3 members after the onset of wound induction. 1
through 5, Wound-treated samples after 0, 0.5, 1, 4, and 12 h of
culture, respectively, in Murashige and Skoog liquid medium; C, the
wound-treated sample after a 4-h culture in distilled water as control.
mains against a given proteinase of external origin
would vary. Evidence showing that effectiveness of
inhibition could vary enormously has been reported
(Belitz et al., 1982; Christeller and Shaw, 1989). Recent data has shown that the effectiveness of inhibitory activity against the same class of proteinase from
the same insect species could vary enormously (Volpicella et al., 2003). Therefore, in response to changes
of proteinases of external origin, an adequate number
of BBI domains with sufficient sequence variations
within the RBBI family would serve as a reservoir
from which more effective inhibitors would evolve
from a process of selection.
In the novel three-domain BBI genes, an additional
domain is present that would contribute to the development of new proteinase-inhibiting activity.
Four reactive-like sites were identified in the F31
protein in this study (Fig. 1E, blue letters in RBBI3-1
sequence), of which two P1 residues are the trypsininhibitory R and K. The other two P1 residues are
two Glu residues (E), whose inhibitory specificity has
not been reported. High trypsin-inhibitory activity
was detected as expected (Fig. 5), indicating that the
two P1 residues function normally. However,
chymotrypsin-inhibitory (Fig. 5), subtilisin-inhibitory,
or papain-inhibitory activity was not detected (data
not shown), suggesting that the two Glu residues in
the P1 sites do not correspond to the proteinaseinhibitory activity we have known and investigated
thus far. The possibility that new inhibitory specificities have evolved in these three-domain inhibitors
needs to be further clarified by biochemical means.
Our detailed northern blotting and in situ hybridization analysis suggested that RBBIs are both developmentally regulated and induced in response to
wound or pathogen attacks. Similar regulatory patterns were found in plant defensive proteins or toxic
secondary compounds (Ryan, 1990; Molyneux and
Ralphs, 1992). The RBBIs of rice are expressed in an
effective and economical way. First, not all of the
members of RBBI genes are expressed equally. The
three-domain genes generally showed higher expression than the two-domain genes (Fig. 3). It is more
likely that the three-domain genes are able to provide
more combinations of different inhibitory activities
when the same amount of protein molecules is synthesized. Second, the expression level of the RBBI
genes is higher in those tissues more likely to experience attack by predators or pathogens, e.g. rehydrated embryo, young leaves, root cap, and flower
organs. It is reasonable to speculate that higher expression of RBBIs in these tissues would serve as an
effective protection mechanism against attacks by
predators or pathogens. One interesting finding is
the detection of high-level expression of RBBIs in
aleurone cells, i.e. expression in the cells surrounding
the endosperm and embryo (Fig. 4, C and D). Moreover, the expression of RBBIs in these tissues is temporally regulated, e.g. the RBBI mRNA level in the
embryo peaked up at 48 h after rehydration (Fig. 4A)
and then dropped precipitously afterward (data not
show). Third, RBBI genes are induced by wound or
pathogen attack (Figs. 6 and 7A). It was recently
reported that the expression of RBBI3-3 could be
induced by cut and jasmonic acid (Rakwal et al.,
2001). It is generally accepted that wounding or attack by predators or pathogens will trigger plant
defense responses that may include inhibitory reactions or even the production of toxic proteins or
chemical compounds (Ryan, 1990; Baldwin and Preston, 1999). On the basis of the three points mentioned
above, it is reasonable to suggest that plants should
have optimized their tradeoffs with respect to the
allocation of resources to growth and development
by choosing an effective way of expressing plant
defense proteins such as BBIs.
The sequence divergence, multiple inhibitory activities, and inducible expression pattern suggest that
RBBIs may serve as potential effective defense proteins in vivo. It has long been known that proteinase
inhibitors could be induced by pathogens or pathoTable I. Resistance test of transgenic rice plants against the rice
fungus pathogen P. oryzae
Pathogen Isolates
cka
T137-2
T191-2
Race 033
96-4-1a
96-4-2a
Race 335 (96-8-1a)
Race 317 (96-10-1a)
Zhong 10-8-14
Sichuan ⫺26
ss
ss
ss
ss
ss
ss
HR
HR
HR
HR
HR
HR
HR
HR
HR
HR
HR
HR
a
ck, Untransformed Taipei 309 as control; ss, highly susceptible
(level 5); HR, highly resistant (level 0).
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Rice Bowman-Birk Inhibitor Gene Family
Figure 7. A, Semiquantitative RT-PCR analysis
of RBBI2-3 expression 2 d after inoculation. C1,
Untransformed rice cv Taipei 309 before inoculation; C2, untransformed rice cv Taipei 309
after inoculation; 1, 2, and 3, three randomly
picked rice plants of T137-2 after inoculation;
1⬘, 2⬘, and 3⬘, three randomly picked rice plants
of T191-2 after inoculation. B, Transgenic rice
cv Taipei 309 plants are resistant to rice blast
fungus pathogens. Non-transformed control
wild-type rice cv Taipei 309 plants were grown
next to the transgenic rice cv Taipei 309 (T137-2
and T191-2) homozygous for the RBBI2-3 transgene. Plants were infected with one of six isolates (Zhong 10-8-14, Sichuan 26, 96-4-1a, 964-2a, 96-8-1a, and 96-10-1a) of P. oryzae. High
resistance of both T137-2 and T191-2 seedlings
was observed against the infection of all of the
six isolates.
gen/plant cell wall-derived elicitors (Roby et al.,
1987; Rickauer et al., 1989; Cordero et al., 1994;
McGurl et al., 1995; Choi et al., 2000; Pernas et al.,
2000). Furthermore, in vitro experiments showed that
growth of fungi or activities of proteinases from
fungi were inhibited by proteinase inhibitors (Senser
et al., 1974; Mosolov et al., 1976, 1979; Brown and
Ryan, 1984; MacGibbon and Mann, 1986; Xie et al.,
1996). Meanwhile, higher levels of trypsin-inhibitory
activity were found in tomato (Lycopersicon esculentum) and wheat varieties with higher resistances
against pathogenic fungi (Peng and Black, 1976; Yamaleev et al., 1980). However, although there is an
increasing amount of direct evidence supporting the
defensive roles of proteinase inhibitors against insects and other herbivores, the role played by pro-
teinase inhibitors in fungal resistance is only supported by indirect evidence (Ryan, 1990).
The transgenic data of this study demonstrates that
proteinase inhibitors play an important role in the
defense response of rice against fungal infection. It is
the first direct evidence that proteinase inhibitors
could arrest fungal invasion and inhibit the growth
of the fungus not only in vitro (Xie et al., 1996) but
also in vivo (Fig. 7B), possibly by inhibiting proteolysis and thereby limiting availability of amino acids.
Our data also showed that the inhibiting effect of
proteinase inhibitor against proteinase in vitro was
dosage dependent. A higher percentage of inhibitory
effect was observed when higher concentrations of
RBBI3-1 were used to incubate with a given concentration of proteinase (Fig. 5A). Although the mRNA
Plant Physiol. Vol. 133, 2003
567
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Copyright © 2003 American Society of Plant Biologists. All rights reserved.
Qu et al.
of RBBI2-3 increased after inoculation in susceptible
plants, more than a 10-fold amount of RBBI2-3
mRNA was found accumulated in transgenic rice
leaves, driven by the 35S promoter (Fig. 7A). This
data suggests that a high concentration of proteinase
inhibitors is required to inhibit growth and multiplication of the pathogens. Pathogenic fungi infect their
host by first secreting hydrolytic enzymes outside of
their bodies to hydrolyze complex substrates into
small organic molecules (Campbell et al., 1999). It is
reasonable to suggest that the overexpression of
RBBI2-3 protein in transgenic rice may inhibit the
function of proteinases secreted by P. oryzae through
a similar biochemical mechanism like RBBI3-1. If different RBBI proteins have different inhibitory activities, it may also be expected that overexpression of
the RBBI member exhibiting the highest inhibitory
activity in transgenic rice would result in the highest
resistance against pathogen.
Two possible signal pathways will be triggered
when a plant is infected by a pathogen: one related to
wound through the penetration and colonization of
the pathogen, and the other through direct molecular
recognition of the pathogen (Cordero et al., 1994).
The fact that RBBI genes in this study are induced by
wounding, together with data from other studies
(Rakwal et al., 2001), suggests that the wound-related
signal pathway is possibly involved in increasing the
expression of RBBIs in rice plants when a pathogen
attacks. Further evidence, however, needs to be
presented.
MATERIALS AND METHODS
Screening of a BAC Library and Analysis of the
Positive Clones
The filters and clones of a rice (Oryza sativa) BAC library, generated from
O. sativa subsp. japonica var Teqing, were kindly provided by Dr. Hongbing
Zhang (University of Texas A & M, College Station). The filters were
immersed in a hybridization buffer (50% [v/v] formamide, 0.12 m Na2HPO4,
pH 7.2, 0.25 m NaCl, and 7% [w/v] SDS). After 30 min of prehybridization
at 43°C, the filters were placed into a fresh hybridization buffer for hybridization. The RBBI8 probe, a fragment size of about 450 bp, was radioactively
labeled with [32P]dCTP (NEN, Boston) using a random primer labeling kit
(Promega, Madison, WI) according to the manufacturer’s instructions. The
probe was denatured by boiling for 2 min and then added to the hybridization buffer. After an 18-h hybridization period at 43°C, the filters were
first rinsed twice with 2⫻ SSC and then washed for 15 min each in 2⫻
SSC/0.1% (w/v) SDS, 0.5⫻ SSC/0.1% (w/v) SDS, and 0.1⫻ SSC/1% (w/v)
SDS at 43°C. The washed filters were exposed and analyzed using a PhosphorImager IS445 (Molecular Dynamics, Sunnyvale, CA). The positive BAC
clone DNA was then digested by EcoRV, HindIII, or XbaI, and a Southern
blot was performed with a Zeta-GT Probe nylon membrane (Bio-Rad, Hercules, CA) using the same probe used to screen the BAC clone library. The
positive DNA fragments were recovered by Qiaquick Gel Extraction Kit
(Qiagen, Valencia, CA) and subcloned into pBlueScript SK⫹ (Stratagene, La
Jolla, CA). The plasmid DNA was purified using the Wizard Plus Minipreps
DNA purification kit (Promega) and sequenced with an ABI 377 DNA
sequencer with a Big Dye Primer Cycle Sequencing Ready Reaction kit
(Applied Biosystems, Foster City, CA). Identified sequences were then
searched for putative ORFs using DNASIS programs (Hitachi, Tokyo). The
deduced amino acid sequences from these putative ORFs were investigated
for similarity to proteins in the EMBL and SWISSPRO databases using the
BLAST search program (Altschul et al., 1997). The amino acid sequence
alignment of rice BBIs was made using ClustalW (Thompson et al., 1994).
The GenBank accession numbers of these genes are as follows: AJ277468
(RBBI2-1, RBBI2-2, RBBI3-1, and RBBI3-2), AJ277469 (RBBI3-3), AJ277470
(RBBI2-3), and AJ277472 (RBBI2-4), respectively.
Northern-Blot Analysis
Total RNA was isolated from rice leaves, roots, flowers, and immature
rice grains either using an RNA Extraction Kit (Amersham Biosciences,
Piscataway, NJ) according to the manufacturer’s instruction or by the guanidium thiocyanate method (Chomczynski and Sacchi, 1987). The total RNA
samples (10 ␮g lane⫺1) were separated on 1% (w/v) agarose gels and were
transferred to a Hybond-N⫹ nylon membrane (Amersham Biosciences).
After a 0.5-h prehybridization, the filters were hybridized in the hybridization buffer (50% [v/v] formamide, 0.12 m Na2HPO4, pH 7.2, 0.25 m NaCl,
and 7% [w/v] SDS) at 43°C for 22 h. Then the filters were first rinsed twice
with 2⫻ SSC and washed for 15 min each in 2⫻ SSC/0.1% (w/v) SDS, 0.5⫻
SSC/0.1% (w/v) SDS, and 0.1⫻ SSC/1% (w/v) SDS at 43°C, respectively.
The insert of prbbi8 (i.e. the coding region of RBBI2-3 and 450 bp in length)
was radioactively labeled with [32P]dCTP (NEN) using a random primer
labeling kit (Promega) according to the manufacturer’s instructions. The
gene-specific primers that are from the 3⬘-UTR sequences of the seven
members of the rice BBI gene family were used to make probes for northern
hybridization. The 3⬘-UTR sequences of RBBIs were amplified by PCR from
the genomic DNA using gene-specific primers (RBBI2-1, 5⬘-ACT GAT TAA
CTA TAG CTA GC-3⬘ and 5⬘-CTA AAG TTG CAC TTT TCT GA-3⬘; RBBI2-2,
5⬘-GAA GAG AAC TAC TTC TCT GT-3⬘ and 5⬘-C TAC CTG CTT TCC ACC
GGA-3⬘; RBBI2-3, 5⬘-CTG CAG ATC GAT ATG TAT GAT-3⬘ and 5⬘-TGA
CTG ATG CGC CTA TGG-3⬘; RBBI2-4, 5⬘-ATG AAT AGC GGC AAT ATG
GC-3⬘ and 5⬘-TCA CTT GTG TTC GTC GGG TG-3⬘; RBBI3-1, 5⬘-TA GCT
ACA GAT GAT CGA TGA-3⬘ and 5⬘-CAA TAT AAG TGT CCT CTC AG-3⬘;
RBBI3-2, 5⬘-TCC TTG CAG ATA TGA TCG AT-3⬘ and 5⬘-AC AAG GTT
AAG GTC AAA CTC-3⬘; and RBBI3-3, 5⬘-GCT ACA GAT GAT CGA TGA
TCG-3⬘ and 5⬘-AC CCA CAA CAT CTT CTA ACG-3⬘). The PCR fragments
were cloned into a pBlueScript vector, and the plasmids were used to make
radioactive probes for the northern-blot filters by PCR. The PCR reactions
containing cDNA: 1⫻ polymerase buffer (Promega); 2.5 mm MgCl2; 200 ␮m
of each of dATP, dGTP, and dTTP; 100 ␮m [32P]dCTP (NEN); 0.1 ␮m of each
primer; and 2 units of TaqDNA polymerase (Promega) were performed in a
final volume of 20 ␮L. The PCR reactions were carried out for 30 cycles
(94°C, 40 s; 58°C, 30 s; and 72°C, 30 s).
In Situ Hybridization
Rice materials including germinating seeds (24, 48, and 72 h after dehydration), 6-d-old seedlings, and 11-d-old seedlings were collected. Rice
tissues were fixed with formaldehyde and were embedded in paraplast. The
tissues were sliced into 5-␮m-thick slices and mounted on poly l-Lys-coated
glass slides. These were then rinsed in 10 mm Tris-HCl, pH 8.0, and
incubated in 1% (w/v) bovine serum albumin in the same buffer for 1 h.
After acetylation, sections were dehydrated with a graded series of ethanol
washes and air-dried. The probe was labeled with digoxigenin (DIG) and
hydrolyzed in hydrolysis carbonate buffer (60 mm Na2CO3 and 40 mm
NaHCO3, pH 10.2) before it was used. After prehybridization, a hybridization solution (0.3 m NaCl, 10 mm Tris-HCl, pH 7.5, 5 mm EDTA, 1⫻
Denhardt’s solution, 50% [v/v] formamide, 2 mg mL⫺1 tRNA, and 200 units
mL⫺1 RNase inhibitor) containing denatured (DIG)-labeled sense or antisense probe -RNA was added. The slides were incubated overnight in a
humid box at 45°C. After two washing steps with 0.2⫻ SSC at 55°C for 30
min each, slides were incubated with 20 ␮g mL⫺1 RNase A (Promega) at
37°C for 30 min, followed by washing with 0.2⫻ SSC at 55°C for 1 h.
Immunological detection of DIG-labeled RNA probe was performed with
anti-DIG antibody conjugated with alkaline phosphatase (Roche Diagnostics, Mannheim, Germany) according to the supplier’s protocol. Staining
was obtained using the substrates nitroblue tetrazolium and 5-bromo-4chloro-3-indolyl phosphate. Slides were analyzed under bright field microscopy (DM RE HC microscope, Leica, Wetzlar, Germany) equipped with a
CCD camera (Eastman Kodak, Rochester, NY). The probe used for the in situ
hybridizations was the conserved coding region of the RBBI2-3 gene (450 bp).
568
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Copyright © 2003 American Society of Plant Biologists. All rights reserved.
Rice Bowman-Birk Inhibitor Gene Family
Constructs for Prokaryotic Expression
The pBS-rbbi21K containing RBBI3-1 was used as the template to amplify
the three-domain fragment. Fifty picomoles each of the 5⬘ primer (5⬘-GGC
GGA TCC GCA CCA CCA CGC CCG CCC-3⬘) and 3⬘ primer (5⬘-TGT AAG
CTT CTA GTT CTC CGC TCG GGG-3⬘), together with 10 units of thermostable pfu DNA polymerase (Stratagene) was used to prime a 100-␮L PCR
(95°C, 1 min; 55°C, 45 s; and 72°C, 1 min, 35 cycles). The amplified 660-bplong fragment that codes for the three BBI domains of RBBI3-1 was purified
and then digested with BamHI and HindIII, which were introduced by the
two primers (bold sequences) before being ligated into pET28a (Novagen,
Madison, WI). Competent cells of Escherichia coli strain BL21 (DE3) were
transformed, and recombinant plasmids were screened and sequenced for
verification. The recombinant plasmid was designated as pET28-F31.
Expression, Refolding, and Purification of the Fusion
Protein F31
The cells were first cultured overnight in 10 mL of Luria-Bertani medium
(50 mg mL⫺1 kanamycin) at 37°C. Five milliliters of the overnight culture
was transferred into 1,000 mL of Luria-Bertani medium and allowed to
continue to grow until A600 reached 0.4 to 0.6. Isopropylthio-␤-galactoside
was added to 0.5 mm and allowed to induce for 3 to 6 h. The bacteria were
harvested and suspended in 50 mL of cell lysis buffer (50 mm Tris-HCl and
100 mm NaCl, pH 8.0) before being sonicated in an ice-water slurry (30 s of
sonication alternated with 30 s of cooling, six times).
Most of the three-domain fusion protein F31 was not soluble in our
experiments, and therefore inclusion bodies were harvested. Collection of
inclusion bodies, protein refolding, and purification were carried out as
described before (Li et al., 1999). The purity of the proteins was checked by
SDS-PAGE.
according to the International Rice Research Institute evaluation standard
(Mackill and Bonman, 1992).
RBBI2-3 Expression Analysis by Semiquantitative
RT-PCR
Total RNA was extracted from leaves of transgenic and control rice plants
48 h after inoculation using the previously described method. After DNase
I treatment, 5 ␮g of the total RNA was used to synthesize the first strand of
cDNA with an oligo(dT) and RT SuperscriptII as recommended by the
manufacturer (Invitrogen, Carlsbad, CA). Two microliters of the first strand
of cDNA was used as a template for semiquantitative RT-PCR. The PCR
reaction containing cDNA 1⫻ polymerase buffer (Promega), 2.5 mm MgCl2,
200 ␮m of each of dNTP, 0.1 ␮m of each primer, and 2 units of TaqDNA
polymerase (Promega) was performed in a final volume of 100 ␮L. Primers
specific for RBBI2-3 are as above, whereas the primers for the internal
control Act 1 gene are 5⬘-CTG ACG GAG CGT GGT TAC TCA TTC-3⬘ and
5⬘-GCT AGG AGC AAG GCA GTG ATC TTC-3⬘. To ensure that the amplification reaction was within linear range, the PCR reactions were carried out
for 20 cycles. Five microliters of the RT-PCR reaction mixtures was loaded
on a 0.8% (w/v) agarose gel, transferred to Hybond N⫹ nylon membranes
(Amersham Biosciences), and hybridized with radioactive-labeled corresponding probes using the procedure detailed previously.
Distribution of Materials
Upon request, all novel materials described in this publication will be
made available in a timely manner for noncommercial research purposes,
subject to the requisite permission from any third-party owners of all or
parts of the material. Obtaining any permissions will be the responsibility of
the requestor.
Inhibitory Activity Assay of the Fusion Proteins F31
Inhibitory activities against trypsin 1:250 (Difco Laboratories, Detroit)
and chymotrypsin (C4129, Sigma-Aldrich, St. Louis) were determined by
incubating purified fusion proteins together with the enzymes at 25°C for 5
min. The remaining trypsin activity was measured with TAME (T4626,
Sigma-Aldrich) as described previously (Hummel 1959). The remaining
chymotrypsin activity was measured with ATEE (A6751, Sigma-Aldrich;
Schwert and Takenaka, 1955). The absorbency at 247 nm (for ATME) and 237
nm (for ATEE) was measured with a GBC Cintra 10e UV-Visible spectrometer (GBC, Melbourne, Australia). Commercial soybean BBI (T9777, SigmaAldrich) was assayed in parallel as positive controls.
ACKNOWLEDGMENTS
We thank Dr. S. McCouch (Cornell University, Ithaca, NY) for help with
the mapping of the marker RBBI8 and Dr. Hongbing Zhang (University of
Texas A & M, College Station) for kindly providing the rice BAC library. We
sincerely thank Prof. XingWang Deng (Yale University, New Haven, CT)
and Dr. Hongwei Guo (University of California, Los Angeles) for critical
review, comments, and valuable discussions and Dr. Matthew Terry (University of Southampton, UK) for help with the manuscript.
Received April 5, 2003; returned for revision April 24, 2003; accepted June 4,
2003.
Wound-Inducibility Analysis
Rice leaves were cut into 0.5-cm2 pieces and placed into an Murashige
and Skoog liquid medium and distilled water for 0.5, 1, 2, 4, and 12 h. RNA
samples were isolated from the treated leaves, and northern blotting was
performed as described.
Rice Transformation, Pathogen Inoculation, and
Resistance Evaluation
Rice cv Taipei 309 was chosen as a host onto which to transform the
RBBI2-3 gene that was cloned into properly digested pMON310 and driven
by the 35S cauliflower mosaic virus promoter. Rice callus was prepared
from immature embryos, and transformation by particle bombardment was
carried out as described previously (Zheng et al., 1997). The regenerated
shoots were selected by streptomycin, and 20 lines of T1 generation seeds
were obtained. The T1 transgenic rice plants were grown to the four-leaf
stage in a greenhouse.
The conidiospores of Pyricularia oryzae were suspended in 0.02% (v/v)
Tween 20 to a concentration of 30 to 50 spores/100⫻ viewing field and were
evenly sprayed onto rice leaves using an N-1 type inoculation device. After
inoculation, the plants were kept at 26°C for 24 h with 100% humidity before
being transferred to a humid greenhouse. The development of disease
symptoms was checked 6 to 7 d later, and the resistance level was evaluated
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