|
Plant Physiol. (1998) 117: 33-41
Molecular Characterization of the Oxalate Oxidase Involved in the
Response of Barley to the Powdery Mildew Fungus1
Fasong Zhou2,
Ziguo Zhang2, 3,
Per L. Gregersen,
Jørn D. Mikkelsen,
Eigil de Neergaard,
David B. Collinge, and
Hans Thordal-Christensen*
Plant Pathology Section, Department of Plant Biology, The Royal
Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871
Frederiksberg C, Copenhagen, Denmark (F.Z., Z.Z., P.L.G., E.d.N.,
D.B.C., H.T.-C.); and Danisco Biotechnology, Copenhagen, Denmark
(J.D.M.)
 |
ABSTRACT |
Previously
we reported that oxalate oxidase activity increases in extracts of
barley (Hordeum vulgare) leaves in response to the
powdery mildew fungus (Blumeria [syn.
Erysiphe] graminis f.sp.
hordei) and proposed this as a source of
H2O2 during plant-pathogen interactions. In
this paper we show that the N terminus of the major pathogen-response
oxalate oxidase has a high degree of sequence identity to previously
characterized germin-like oxalate oxidases. Two cDNAs were isolated,
pHvOxOa, which represents this major enzyme,
and pHvOxOb', representing a closely related
enzyme. Our data suggest the presence of only two oxalate oxidase genes
in the barley genome, i.e. a gene encoding
HvOxOa, which possibly exists in several
copies, and a single-copy gene encoding HvOxOb. The use of 3 end gene-specific probes has allowed us to demonstrate that the HvOxOa transcript accumulates to 6 times the level of the HvOxOb transcript in
response to the powdery mildew fungus. The transcripts were detected in
both compatible and incompatible interactions with a similar
accumulation pattern. The oxalate oxidase is found exclusively in the
leaf mesophyll, where it is cell wall located. A model for a signal
transduction pathway in which oxalate oxidase plays a central role is
proposed for the regulation of the hypersensitive response.
| |
INTRODUCTION |
The generation of AOS has been recognized as a significant
phenomenon during pathogen-plant interactions (Baker and Orlandi, 1995 ;
Low and Merida, 1996). AOS have been suggested to be involved in plant
defense responses in several ways: in cross-linking of lignin and
proteins during cell wall modification; in signal transduction leading
to gene regulation, hypersensitive cell death, and systemic acquired
resistance; and as antimicrobial agents, which inhibit pathogen
development directly.
We demonstrated recently that
H2O2 accumulates at the
sites of contact between epidermal cells undergoing an HR and the
subjacent mesophyll cells (Thordal-Christensen et al., 1997). Several
possible sources of defense-related AOS have been suggested, one of
which is the
H2O2-generating oxalate
oxidase. Increased activity of this enzyme is found in extracts of
barley (Hordeum vulgare) and wheat (Triticum
aestivum) leaves following inoculation with the powdery mildew
fungus (Blumeria [syn. Erysiphe]
graminis; Dumas et al., 1995; Zhang et al., 1995; Hurkman
and Tanaka, 1996b). Oxalate oxidases are known from a number of plant,
fungal, and bacterial species (Pundir, 1991). Of these, only the wheat
and barley enzymes have been classified as germin-like oxalate oxidases (Dumas et al., 1993; Lane et al., 1993).
The germin-like oxalate oxidases are homo-oligomeric, water-soluble,
heat-stable, protease-resistant, SDS-tolerant glycoproteins originally
known to be expressed in cell walls of cereal embryos at the onset of
germination (Lane, 1994). In total, six potentially different oxalate
oxidase and oxalate oxidase-like proteins have been characterized in
barley, either based on amino acid or cDNA sequence: two salt-response
proteins from roots (Hurkman et al., 1991), a root cDNA/protein (Lane
et al., 1993), a root cDNA (Hurkman et al., 1994), a seedling protein
(Dumas et al., 1993), and a leaf epidermal cDNA (Wei et al., 1998).
Only the root protein of Lane et al. (1993) and the seedling protein of
Dumas et al. (1993) have been demonstrated to possess oxalate oxidase
activity.
The pathogen-response oxalate oxidase in barley expressed in response
to the powdery mildew fungus (Dumas et al., 1995; Zhang et al., 1995)
appears as two bands on SDS-PAGE as oligomers of approximately 95 and
100 kD. Both are serologically related to the germin-like oxalate
oxidase of wheat (Zhang et al., 1995). In the present study, we present
the N-terminal sequence from the 100-kD oligomer and its corresponding
cDNA. A highly related cDNA is also presented, and these two clones
represent the two oxalate oxidase genes apparently present in barley.
Both genes express transcripts that accumulate following attack by the
powdery mildew fungus. Pathogen-response oxalate oxidase is found to be strictly confined to the leaf mesophyll and may play a role in a signal
transduction pathway for regulation of the HR.
| |
MATERIALS AND METHODS |
Two near-isogenic lines of barley (Hordeum vulgare L. cv Pallas), P-01 and P-02, were grown in a growth chamber under 16 h of light (100 mE s 1
m2) at 60% RH and 20°C and 8 h of dark
at 80% RH and 5°C. Isolates C15 and A6 of the powdery mildew fungus
(Blumeria [syn. Erysiphe] graminis
f.sp. hordei; Bgh) were used for uniform,
high-density (100-200 conidia/mm2) inoculation
of the abaxial epidermis of first leaves, as described previously
(Thordal-Christensen and Smedegaard-Petersen, 1988). P-01 and P-02
possess the Ml-a1 and Ml-a3 resistance genes to Bgh, respectively (Kølster et al., 1986). P-01 exhibits
single-cell HR resistance to C15 and is susceptible to A6. P-02 is
susceptible to C15 and exhibits a multicell HR resistance to A6.
Irrespective of these outcomes of the different interactions, papilla
formation always arrests a high percentage of the fungal conidia at the stage of penetration (Thordal-Christensen and Smedegaard-Petersen, 1988).
Epidermal and mesophyll (including the noninoculated epidermis) tissues
were separated by making an incision near the leaf tip, and the
abaxial, inoculated epidermis was then stripped off. For whole-leaf
samples, approximately 6 cm of the central part of the leaf was
collected.
Protein Purification, Preparation of Antibodies, and Amino Acid
Sequencing
Oxalate oxidase was extracted and partially purified from infected
leaves (P-01/A6, harvested 6 d after inoculation) according to the
method of Zhang et al. (1996). Following preparative 6% SDS-PAGE
(without prior boiling in reducing agent: oxalate oxidases are SDS
tolerant), the gel strip containing the 100- and 95-kD oxalate oxidase
proteins was used for immunizing a rabbit according to the method of
Harlow and Lane (1988). Fifty micrograms of protein was injected seven
times. In addition to recognizing the native 100- and 95-kD oxalate
oxidase proteins, the obtained antiserum recognized numerous barley
leaf proteins.
To immunopurify a specific oxalate oxidase antibody, the mature oxalate
oxidase polypeptide with six His residues fused to the N terminus was
expressed in Escherichia coli. This was accomplished by
ligating a PCR-amplified fragment of pHvOxOa into the
expression vector pQE-9 (Diagen, Hilden, Germany). Expression of the
protein was induced, and the protein was purified subsequently on an
Ni2+ column under denaturing conditions according
to the recommendations of the manufacturer. This purified, denatured
monomer was then dotted onto a nitrocellulose membrane. The membrane
was dried and used to purify the oxalate oxidase-specific antibody. The membrane was blocked with 4% BSA and then washed with 0.1 m Gly-HCl, pH 2.8, for 5 min. After the membrane was
incubated overnight with the antiserum and extensive washing with 1×
TBS, purified antibody was eluted from the membrane with 0.1 m Gly-HCl, pH 2.8, for 2 min. The buffer with purified
antibody was quickly transferred to 0.03 volume of 1 m
Tris-base, thereby adjusting the pH to 7.0. The purified antibody
specifically recognized oxalate oxidase. With approximately the same
activity, it recognized two bands of the native (Fig. 7; see below) and
one band of the denatured oxalate oxidase (data not shown). The native
bands were confirmed by activity staining in an identical SDS gel (Fig.
7). Alternatively, the 100-kD oxalate oxidase was blotted onto a PVDF
membrane and subjected to amino acid sequencing according to the method
of Nielsen et al. (1993).
View larger version (66K):
[in this window]
[in a new window]
| Figure 7.
Tissue localization of the pathogen-response
oxalate oxidase in barley leaves (P-01, 24 h after inoculation
with Bgh [C15]) demonstrated on 12% SDS-PAGE gels
using samples of the whole leaf (lanes 1), epidermal tissue (lanes 2),
and mesophyll tissue (lanes 3). Twenty microliters of protein extract
(representing equal amounts of fresh weight tissue) was loaded in each
lane as unboiled samples using a loading buffer lacking reducing agent.
A, Silver-stained gel. B, Immunoblot incubated with purified oxalate
oxidase antibody. C, In-gel oxalate oxidase activity assay.
|
|
Plaque, Northern-, and Southern-Blot Hybridization
Plaque lifts from a  -cDNA library were prepared on
nitrocellulose (Millipore) essentially according to the method of
Sambrook et al. (1989). Northern blots on Zetaprobe membranes (Bio-Rad) and genomic Southern blots on Hybond N+ membranes
(Amersham) were prepared essentially according to the method of
Sambrook et al. (1989). Even loading of northern blots was confirmed by
methylene blue (0.02%) staining of rRNA on the blot prior to
hybridization. 32P-labeled probes of insert DNA
were prepared using the Megaprime DNA labeling system (Amersham). PCR
fragments for gene-specific probes were synthesized using the KS primer
(Stratagene) and 5-CTTAACTTCCATGAGCCC-3 on pHvOxOa,
and the SK primer (Stratagene) and 5-AATTCCTGGGAGCCTTC-3 on
pHvOxOb '. 32P-labeled
gene-specific probes were subsequently prepared using the Megaprime DNA
labeling system with the same two sets of primers replacing the random
primers. Hybridizations of plaque lifts and northern blots were
performed according to the method of Bryngelsson et al. (1994).
Hybridizations of genomic Southern blots were performed in the aqueous
solution described by Anderson and Young (1985). See figure legends
for wash stringency conditions. Relative hybridization signals were
determined by contour densitometry using ImageMaster1D software
(Pharmacia).
RNA and DNA Extraction and Molecular Techniques
Total RNA was obtained from frozen leaf material essentially
according to the method of Collinge et al. (1987). Barley genomic DNA
was isolated according to the method of Ausubel et al. (1987). Selected
cDNA clones of a -ZAPII library were converted to pBluescriptII SK( ) plasmid clones by the in vivo excision procedure recommended by
the manufacturer (Stratagene).
DNA sequencing was performed using the Sequenase DNA sequencing kit
(United States Biochemical) according to the manufacturer's recommendations, using [35S]-dATP (Amersham).
The reactions were resolved on 8 m urea, 6% polyacrylamide
buffer gradient (1-5× TBE) gels. The nucleotide sequences were
determined in both orientations using the KS, SK, T3, and T7 primers on
double-stranded plasmid DNA of the presented clones and derived
subclones, all in the pBluescriptII SK() plasmid vector.
Computer-assisted analysis of sequence data was performed using DNASIS
software (version 5.02, Pharmacia) and the GCG (Genetics Computer
Group, Madison, WI) package (Devereux et al., 1984). The sequence
databases provided by EMBL and GenBank were searched for related
sequences using the GCG package.
Protein Electrophoresis, Immunoblotting, and Activity Assay
Water extracts of frozen tissue powders (2 g fresh tissue/mL) were
loaded onto SDS-PAGE in a loading buffer lacking reducing agent and
without boiling (Sambrook et al., 1989). Proteins were blotted onto
nitrocellulose (Bio-Rad) in a semidry blotter according to the method
of Harlow and Lane (1988). Subsequent immunodetection of proteins on
the blot was performed according to standard procedures. Alkaline
phosphatase-conjugated goat anti-rabbit antibody (Sigma) was used as a
secondary antibody. Oxalate oxidase active proteins were revealed by
the in-gel assay described by Zhang et al. (1996). Rainbow
Mr standards (Amersham) were used.
In Situ Detection of Oxalate Oxidase Activity
Approximately 3 × 5-mm2 leaf specimens
of 8-d-old P-02 plants, harvested 24 h after inoculation with C15,
were incubated at room temperature in an oxalate oxidase activity
developer solution (40 mm succinic acid/NaOH, pH 3.5, 2 mm oxalic acid, 0.5 mg/mL 4-chloro-4-naphthol, and 3.5 mm EDTA) adapted from that of Dumas et al. (1995) for in
situ activity detection. Very clear (positive) dark-blue staining
appeared in the specimens after 4 h of incubation. Specimens
incubated in developer solution lacking oxalic acid were used as
activity-staining controls. The stained specimens were fixed in 4%
paraformaldehyde in PBS (130 mm NaCl, 7 mm
Na2HPO4, and 3 mm NaH2PO4, pH
7.0). After being washed in PBS, the specimens were infiltrated in a
series of gelatin solutions (5-20%) in PBS at 40°C and embedded in
20% gelatin. The blocks were frozen to 20°C and stabilized with
ice, and 30-µm sections were made by cryostat-sectioning in a rotary
retracting microtome (model 5030, Bright, Huntingdon, UK). Sections
were examined by light microscopy and photographed.
| |
RESULTS |
N-Terminal Amino Acid Sequence
We (Zhang et al., 1995) and Dumas et al. (1995) have showed
previously that there is a significant elevation of the oxalate oxidase
activity in extracts of barley leaves in response to inoculation with
the powdery mildew fungus. The oligomer of this pathogen-response oxalate oxidase appears as activity bands and as immunobands of 95 and
100 kD, respectively, on SDS-PAGE (Zhang et al., 1995; Fig. 7). The
major 100-kD isoform, which will be designated here as
HvOxOa for H.
vulgare oxalate
oxidase a, was purified and denatured to its
monomeric form for N-terminal sequencing. A sequence of 30(28) amino
acids was obtained, demonstrating a 93 to 100% identity with published
barley oxalate oxidases (Fig. 1).
View larger version (8K):
[in this window]
[in a new window]
| Figure 1.
N-terminal amino acid sequence of the monomer of
the 100-kD oligomeric oxalate oxidase HvOxOa.
Alignment to the deduced amino acid sequences of the cDNA clones
pHvOxO-Lane (Lane et al., 1993) and
pHvOxOb (Hurkman et al., 1994) and to the N-terminal
amino acid sequence of HvOxO-Dumas (Dumas et al.,
1993) are shown. Dots indicate amino acids also found in
HvOxOa; X, amino acid not determined.
|
|
Isolation and Characterization of Oxalate Oxidase cDNA Clones
Two barley root oxalate oxidase cDNA clones have been reported
(Lane et al., 1993; Hurkman et al., 1994), and a comparison between our
N-terminal amino acid sequence and the polypeptides encoded by these
two cDNAs suggests that the pathogen-response oxalate oxidase
transcript will be nearly identical to these (Fig. 1). Hybridization of
the cDNA pHvOxO-Lane of Lane et al. (1993) to blots
of RNA extracted from barley leaves following inoculation with the
powdery mildew fungus indicated expression of a pathogen-response oxalate oxidase transcript (Fig. 2).
Therefore, a cDNA library was screened to clone and characterize
this pathogen-response oxalate oxidase transcript. A cDNA library (no.
2) of Thordal-Christensen et al. (1992), prepared from barley (P-01)
leaf poly(A+) RNA extracted 6 h after
inoculation of 10-d-old plants (Thordal-Christensen et al., 1992), was
screened using pHvOxO-Lane. The northern blot data
in Figure 2 suggest that pathogen-response oxalate oxidase cDNAs should
be present in this library. One positive clone was identified among
approximately 6000 plaques. Partial sequence analysis suggested that
this cDNA represents a full-length oxalate oxidase transcript. This
clone was used as a probe to screen another 6500 plaques, and two
additional (a full-length and a partial) oxalate oxidase cDNA clones
were isolated.
View larger version (50K):
[in this window]
[in a new window]
| Figure 2.
Accumulation of oxalate oxidase transcripts in
barley leaves inoculated with the powdery mildew fungus. Total RNA was
extracted from P-01 and P-02 barley leaves following inoculation with
isolate C15 of Bgh (+) and from noninoculated control
leaves (). The transcripts were detected on northern blots by
hybridization with the pHvOxO-Lane cDNA (Lane et
al., 1993). Low- to medium-stringency wash was with 2× SSC at
68°C.
|
|
Complete sequencing demonstrated that the two full-length clones
exhibit a high nucleotide identity. They were subsequently named
pHvOxOa and pHvOxOb' (see also
below). The partial cDNA clone is identical to the 3 end of
pHvOxOa. pHvOxOa is 970 bp and
pHvOxOb' is 954 bp in length. Comparison with the
reported barley oxalate oxidase cDNA sequences showed that
pHvOxOa has a nucleotide identity of 98% with
pHvOxO-Lane (Fig. 3).
However, because the cDNA of Lane et al. (1993) covers only the
sequence encoding the mature protein (the region between positions 164 and 766 of pHvOxOa), the sequence analysis cannot
determine unambiguously that these two cDNAs represent copies of the
same gene rather than individual members of the gene family (see also
below). pHvOxOb' is identical to the region between
positions 28 and 981 of the cDNA of Hurkman et al. (1994; Fig. 3).
We will therefore refer to this latter cDNA in the following
description, and we propose to name it pHvOxOb.
pHvOxOa and pHvOxOb both
contain open reading frames of 672 bp, encoding polypeptides of 224 amino acids (Fig. 3). The overall identity between
pHvOxOa and pHvOxOb is 84%, the main differences occurring outside the reading frame. In the coding regions, the two transcripts exhibit a nucleotide identity of 90%. In
the 5 UTR, the nucleotide identity is 71%, whereas in the 3 UTR, it
is 70% (Fig. 3). We therefore consider these two clones to represent
individual members of the oxalate oxidase gene family (see also
below).
View larger version (60K):
[in this window]
[in a new window]
| Figure 3.
Nucleotide sequence of
pHvOxOa. Alignment to the oxalate oxidase
encoding cDNA clones pHvOxO-Lane (Lane et al.,
1993) and pHvOxOb (Hurkman et al., 1994) are
shown. Dashes indicate introduced gaps; dots indicate nucleotides also
found in pHvOxOa. Start and stop codons are in
bold. Underlined 3 sequences represent segments used as gene-specific
probes.
|
|
Amino Acid Sequence Comparison of Barley Oxalate Oxidases
The N-terminal sequence of 30(28) amino acids of
HvOxOa purified from the inoculated barley leaves
matched perfectly the pHvOxOa-encoded polypeptide
from its amino acid no. 24 (Fig. 4). This
suggests that the immature protein has a 23-amino acid leader sequence, which causes the mature protein to be synthesized into the ER, and to
be potentially exported to the apoplast. This 23-amino acid sequence
apparently has the required lipophilic nature. Furthermore, a cleavage
site at this position was found in all N-terminal sequence analyses of
related barley proteins (Fig. 4). After cleavage, the molecular masses
of HvOxOa and HvOxOb, the deduced
polypeptides of pHvOxOa and
pHvOxOb, were 21.3 and 21.1 kD, respectively.
Alignment of the polypeptide encoded by pHvOxOa, the
30(28) amino acids of HvOxOa, and the 52(51) amino
acids of the three sequences of Dumas et al. (1993) showed 100%
identity. We therefore infer these to be products of the same gene.
Within the 52(51) amino acids of the sequences of Dumas et al. (1993),
one (the N-terminal) differed in relation to the deduced polypeptide of
pHvOxO-Lane and three (including the N-terminal)
differed in relation to HvOxOb. This further supports
our assumption that the polypeptide of Dumas et al. (1993) and the
pHvOxOa-encoded polypeptide are identical. Only 3 of
201 amino acids differed between the polypeptides encoded by
pHvOxOa and pHvOxO-Lane, which
possibly represent gene copies (Fig. 4). The mature polypeptides,
HvOxOa and HvOxOb, have an amino
acid identity of 95%.
View larger version (24K):
[in this window]
[in a new window]
| Figure 4.
Amino acid sequence encoded by the
pHvOxOa cDNA. Alignment to the deduced amino acid
sequences of the cDNA clones pHvOxO-Lane (Lane et
al., 1993) and pHvOxOb (Hurkman et al., 1994) and
to the amino acid sequences of HvOxO-Dumas (Dumas
et al., 1993) and of Gs1 and Gs2 (Hurkman et al., 1991). Lowercase
letters indicate leader sequences; dots represent amino acids also
found in HvOxOa. Identity (%), Amino acid
identity between a polypeptide and the mature
HvOxOa.
|
|
Which Oxalate Oxidase Transcript Is Pathogen Responsive?
When either pHvOxOa or
pHvOxOb' was used to probe northern blots of RNA
samples from inoculated barley leaves, the same accumulation pattern
shown in Figure 2 was obtained (data not shown). We investigated whether this was the result of cross-hybridization between the highly
similar (84%) nucleotide sequences or the presence of two pathogen-response transcripts by using gene-specific probes generated from the relatively divergent 3 UTRs of pHvOxOa and
pHvOxOb' (underlined in Fig. 3). These two 3 probes
were hybridized to a set of identical northern blots with the RNA
samples also used in Figure 2 (Fig. 5).
After parallel hybridization with equally labeled probes, a parallel
high-stringency wash (0.1× SSC, 68°C), and parallel exposure and
film development, indistinguishable transcript accumulation patterns
were resolved. However, the signal obtained with
pHvOxOa 3 probe was generally 6-fold stronger as determined by densitometry. The specificity of the two probes appears
sufficient for distinguishing the individual transcripts as evaluated
by the almost complete lack of cross-hybridization to the other cDNA
clone present on the northern blot (Fig. 5). Note also that
hybridizations using the same 3 probes to genomic Southern blots were
gene specific after only a medium-stringency wash (see below). These
results strongly suggest that the HvOxOa transcript
is pathogen responsive, whereas some doubt remains in relation to the
HvOxOb transcript. However, the weak but clear signal
with the pHvOxOb' 3 probe, our isolation of the
pHvOxOb' cDNA from this particular library, and the
general lack of oxalate oxidase transcript signal in the noninoculated
control samples suggest that the HvOxOb transcript is
pathogen responsive as well, albeit at a very low level.
View larger version (51K):
[in this window]
[in a new window]
| Figure 5.
HvOxOa and HvOxOb
transcripts accumulate in barley leaves inoculated with the powdery
mildew fungus. Total RNA samples of Figure 2 were applied. The
transcripts were detected on northern blots by parallel hybridization
with the gene-specific 3 UTR probes pHvOxOa-3-UTR (A) and
pHvOxOb'-3-UTR (B). The degree of cross-hybridization determined on
plasmid DNA was dotted onto the northern blot. High-stringency washing
was with 0.1× SSC at 68°C. Lanes +, Inoculated; lanes ,
noninoculated.
|
|
Detailed Accumulation Patterns of the Oxalate Oxidase
Transcripts in Different Barley-Powdery Mildew Interactions
The accumulation patterns of the pathogen-response oxalate oxidase
transcripts during plant-pathogen interactions were studied in a
detailed time-course experiment of barley exhibiting different interaction phenotypes to the powdery mildew fungus (Fig.
6). Barley line P-01 exhibited a single
cell HR to isolate C15. P-02 exhibited a multicell HR to isolate A6,
whereas it was susceptible to isolate C15. All interactions were
characterized by a high rate of papilla formation, which arrests
approximately 90 to 95% of all penetration attempts. Northern
hybridization was performed using the full-length
pHvOxOa. This probe will hybridize to both the
HvOxOa and the HvOxOb transcripts;
however, the patterns of accumulation appear the same, the only
difference being in amount (see above). The hybridization showed
accumulation of oxalate oxidase transcripts in the leaves in both
compatible and incompatible interactions (Fig. 6). A very small amount
of oxalate oxidase transcript appeared in noninoculated control leaves.
Transcript accumulation was obvious from 6 h after inoculation in
all interactions. The accumulation patterns differed slightly between
compatible and incompatible interactions. In the P01/C15 and P02/A6
(both incompatible) interactions, one peak of transcript accumulation was detected at 15 and 24 h after inoculation, respectively. In the P02/C15 (compatible) interaction, there were two peaks. One appeared at 15 h and the other at 96 h. The accumulation
profiles agree well with those shown in Figures 2 and 5.
View larger version (124K):
[in this window]
[in a new window]
| Figure 6.
Detailed expression pattern of oxalate oxidase
transcripts demonstrated on northern blots of total RNA extracted from
P-01 and P-02 barley leaves following inoculation with isolates A6 and
C15 of Bgh. The transcripts were detected by
hybridization with the pHvOxOa cDNA.
High-stringency washing was with 0.1× SSC at 68°C.
|
|
Oxalate Oxidase Localization in Leaf Tissue
Thordal-Christensen et al. (1997) detected
H2O2 generation in the
inoculated barley leaves undergoing HR by the use of a
3,3-diaminobenzidine-uptake method.
H2O2 appeared in the
mesophyll tissue under epidermal cells undergoing HR. The staining
commenced in a few of the mesophyll cells attached to the epidermal HR
cell and then extended to all mesophyll cells under the HR cell. If
oxalate oxidase is responsible for the
H2O2 generation under
epidermal HR cells, the enzyme activity should be detectable in
mesophyll tissue of inoculated barley leaves. Protein extracts from
different parts of inoculated leaves were studied with an in-gel
activity assay and an oxalate oxidase antibody. Figure
7 shows that the pathogen-response
oxalate oxidase occurred specifically in the mesophyll tissue, not in
epidermal tissue. Note that the amount of protein loaded onto each lane represents the same amount of fresh weight tissue and that the epidermal tissue constitutes only approximately one-tenth of the whole
leaf. Therefore, the epidermal sample represents 10 times as much leaf
area. Mesophyll location has previously been determined at the
transcript level (Gregersen et al., 1997).
Cross-sections of leaves stained for oxalate oxidase activity
further confirm the mesophyll location of the pathogen-responsive oxalate oxidase. Leaf segments collected 24 h after inoculation gave a only a very faint response in the chloroplasts when oxalic acid
was omitted from a developer solution adapted from Dumas et al. (1995;
Fig. 8A). On the other hand, a very
strong dark-blue staining reaction was observed after incubation in the
developer solution containing oxalic acid (Fig. 8B). That this
H2O2-requiring staining
reaction is dependent on oxalic acid strongly indicates specificity for
oxalate oxidase. The faint response in the chloroplasts is likely to be
due to an independent generation of AOS in these organelles. The oxalic
acid-dependent staining reaction occurred throughout the entire
mesophyll, where it was located at the margin of the cells (Fig. 8B). A
very strong response occurred in the vascular tissue, especially in the
phloem tissue and various types of parenchyma (Fig. 8C). The resolution
of the light micrograph was not sufficient to determine whether the
epidermal cell walls facing the mesophyll were stained; however, the
extract of epidermal strips, which contains this particular cell wall,
did not contain oxalate oxidase (Fig. 7).
View larger version (56K):
[in this window]
[in a new window]
| Figure 8.
Cross-sections of oxalate oxidase activity-stained
barley leaves (P-02, 24 h after inoculation with
Bgh [C15]). The staining reaction was performed
without (A) and with (B and C) oxalate. Images are of epidermis
(top)/mesophyll section (A and B) and vascular bundle (C). e, Boundary
between epidermal cells; m, boundary between mesophyll cells. Bar
represents 10 µm on all images.
|
|
Genomic Organization of Oxalate Oxidase Genes
Information concerning the barley oxalate oxidase genes was
provided by a set of identical genomic Southern blots following hybridization with pHvOxOa and
pHvOxOb' (Fig. 9, A
and B). Nearly identical and rather simple band patterns were obtained
with these two probes following a low- to medium-stringency wash. The
specific identity of the bands was easily unravelled by hybridization
using the 3 UTRs (Fig. 9, C and D). This identification is partially confirmed in the relative intensity of the bands obtained after hybridization using the full-length cDNAs. However,
pHvOxOa-specific bands are relatively strong in the
pHvOxOb' hybridization, which is compatible with
the interpretation that the gene for HvOxOa exists in
more than one copy. It appears that there are no introns in the open
reading frames of the genes for HvOxOa and
HvOxOb. This is clear from the result in the
HindIII lane, in which a single prominent band of
approximately 0.8 kb was found for both of the full-length cDNAs. This
band reflected an internal, approximately 0.8-kb HindIII
fragment in both cDNAs (Fig. 3). Therefore, the approximately 0.8-kb
HindIII band in Figure 9, A and B, must be at least a
double band.
View larger version (106K):
[in this window]
[in a new window]
| Figure 9.
Southern blot of barley (P-01) genomic DNA
hybridized with full-length cDNA clones (A and B) and with
gene-specific 3 UTRs (C and D). Low-/medium-stringency washes with 2×
SSC at 68°C (A and B), and medium-stringency washes with 1× SSC at
68°C (C and D).
|
|
The parts of the genes located outside the HindIII fragments
were necessarily accounted for by the very faint bands that were dissimilar for the pHvOxOa and
pHvOxOb' probes, i.e. approximately 1.4 and 2.2 kb,
respectively, for pHvOxOa and approximately 6.0 kb for pHvOxOb' (only one such band could be
detected with this probe). A potential third, less-related gene was
represented by the approximately 1.6-kb HindIII fragment in
Figure 9, A and B. This potential third gene was possibly also
represented by the unidentified bands in the remaining restriction
enzyme lanes (Fig. 9, A and B).
| |
DISCUSSION |
In previous reports of elevated levels of oxalate oxidase activity
in barley-powdery mildew interactions (Dumas et al., 1995; Zhang et
al., 1995), as well as of oxalate oxidase transcript accumulation
induced by B. graminis in wheat (Hurkman and Tanaka, 1996b),
no attempts were made to identify the specific pathogen response
isoenzyme/transcript species. Here we report a barley cDNA
(pHvOxOa) representing a pathogen-response oxalate
oxidase transcript. We found that the cDNA represents a pathogen
response transcript based on N-terminal amino acid sequencing of the
pathogen-response oxalate oxidase, based on northern-blot hybridization
with 3 UTRs and on the fact that pHvOxOa was
isolated from a library prepared from plant material following B. graminis inoculation. The closely related HvOxOb
transcript is also suggested to be pathogen responsive, albeit at a
very low level. The HvOxOb enzyme has not been
identified in extracts following inoculation.
Our data suggest the presence of two germin-like oxalate oxidases
in barley. In the present study we identified cDNAs for both
pHvOxOa and pHvOxOb'. Based on a
coding sequence nucleotide identity of 98%, we suggest that a partial
cDNA identified by Lane at al. (1993) and pHvOxOa
represents copies of the same genes. A cDNA identified by Hurkman et
al. (1994) is 100% identical to but longer than
pHvOxOb', and therefore we propose to designate that
clone pHvOxOb. The overall nucleotide identity
between pHvOxOa and pHvOxOb is
84%. The presence of two germin-like oxalate oxidases in barley is
also supported by the results of genomic Southern analysis presented
here. pHvOxOa and pHvOxOb' clearly
hybridize to different restriction fragments, and since no other
dominant bands can be detected, we conclude that no other gene members of this close relationship exist in barley. However, more distantly related members of the germin-like oxalate oxidase family do exist.
Hurkman et al. (1991) presented 13(12) amino acid N-terminal sequences
of two salt-response root proteins that show 77 and 85% identity to
the oxalate oxidases described here (Fig. 4). Whether these root
proteins have oxalate oxidase activity is not known. In addition, a
multicopy gene that encodes an oxalate oxidase-like protein,
HvOxOLP, with only 46% amino acid identity to
HvOxOa and HvOxOb, has been
identified by Wei et al. (1998). This protein, which seemingly exhibits
no oxalate oxidase activity, is also pathogen responsive.
HvOxOLP is exclusively expressed in the epidermis, in
contrast to HvOxOa. In summary, the germin-like
oxalate oxidase family appears to consist of several closely and more
distantly related members, and the matter is further complicated by the fact that certain members are encoded by genes of a few to many copies.
No amino acid sequence was obtained from the 95-kD pathogen-response oxalate oxidase. However, we speculate that this protein may be HvOxOb. Alternatively, it may represent a
posttranslational modification of the 100-kD HvOxOa.
This is based on the fact that expression of a single wheat oxalate
oxidase gene in tobacco gave rise to a double protein band (Berna and
Bernier, 1997).
Oxalate oxidase transcript (this study) as well as enzyme (Zhang et
al., 1995) accumulates in both compatible and incompatible interactions. The transcript is detected 6 h after inoculation, whereas the enzyme is detected 15 to 24 h after inoculation.
However, while the transcript level declines (particularly rapidly in
the incompatible interactions), the enzyme level remains high, which presumably reflects the unusual stability of this enzyme. In this context, it is of interest that the relative level of the oxalate oxidase transcript is very low compared with other pathogen response transcripts. For instance, its level is only approximately 5% of the
chitinase transcript (Gregersen et al., 1997).
Oxalate oxidase accumulates in barley seedlings upon onset of
germination (Dumas et al., 1993, 1995). The transcript accumulates to
high levels in barley roots upon germination and after treatment with
NaCl, ABA, methyl salicylate, ABA, and IAA, as demonstrated by
hybridization using what we now call pHvOxOb,
followed by a high-stringency wash (Hurkman and Tanaka, 1996a). To
understand the gene regulation, it is of interest to know the extent to
which those signals are due to the HvOxOa and the
HvOxOb transcripts.
We have localized the pathogen-response oxalate oxidase as being evenly
distributed throughout the mesophyll tissue. In situ activity staining,
which suggests that the enzyme is located at the margin of the
mesophyll cell, the fact that the immature protein has a leader
sequence, and the failure of attempts to wash the enzyme out of the
leaf intercellular space (data not shown) suggest that this
water-soluble protein is trapped in the mesophyll cell walls. Such a
location suggests the enzyme to be a possible source for the
H2O2 observed in the zones
of attachment between an epidermal cell undergoing HR and subjacent
mesophyll cells (Thordal-Christensen et al., 1997).
A potential role of this
H2O2 in signaling for HR
will require a specific posttranslation activation of oxalate oxidase
in the incompatible interaction. Oxalate oxidase has maximum activity at pH 3.2 (Sugiura et al., 1979) and will be inactive at the
intercellular pH of 5.5 to 6.0 in a nonstressed leaf. We therefore
hypothesize that signaling for HR involves acidification of the
apoplast under epidermal cells determined to undergo HR. An increase in
the plasma membrane H+-ATPase of such epidermal
cells will potentially lead to an activation of the pathogen-response
oxalate oxidase in the mesophyll cell wall, partly because of a
more suitable pH but also because the substrate, oxalate, otherwise
will be inaccessible in salts of divalent cations.
Pathogen-elicitor-stimulated plasma membrane H+-ATPase activity has been reported in barley
(Wevelsiep et al., 1993; Knogge, 1996) and tomato (Vera-Estrella et
al., 1994; Xing et al., 1996). In the tomato system, stimulation was
found only in incompatible interactions. As indicated above, a decrease
in extracellular pH will not only solubilize oxalate; it will also increase the free Ca2+ concentration (Trewavas
and Gilroy, 1991). H2O2 and
Ca2+ have been suggested to be involved in
signaling for HR (Levine et al., 1996; Price et al., 1996). We are
currently attempting to resolve such an array of HR-signaling events in
which oxalate oxidase potentially plays a central role.
| |
FOOTNOTES |
1
This study was supported by the Danish
Agricultural and Veterinary Research Council, by the Daloon Foundation,
Denmark (F.Z.), and by the Carlsberg Foundation (H.T.-C.).
2
Permanent address: Department of Agronomy,
Huazhong Agricultural University, Wuhan, People's Republic of China.
3
Present address: Department of Plant Sciences,
University of Oxford, UK.
*
Corresponding author; e-mail htc{at}kvl.dk; fax
45-35-28- 33-10.
Received July 29, 1997;
accepted February 4, 1998.
The nucleotide sequence of pHvOxOa reported in
this paper appears in the EMBL nucleotide database under the accession
number Y14203.
| |
ABBREVIATIONS |
Abbreviations:
AOS, active oxygen species.
HR, hypersensitive
response.
UTR, untranslated region.
| |
ACKNOWLEDGMENTS |
We are indebted to Professor Jim Dunwell (University of Reading,
UK; formerly of Zeneca Seeds, UK) for providing a barley oxalate
oxidase cDNA clone.
| |
LITERATURE CITED |
Anderson MLM,
Young BD
(1985)
Quantitative filter hybridisation.
In
BD Hames,
SJ Higgins,
eds, Nucleic Acid Hybridisation A Practical Approach.
IRL Press, Oxford, UK, pp 73-111
Ausubel FM, Brent R, Kingston RE, Moore DD, Smith JA, Seidman JG,
Struhl K (1987) Current Protocols in Molecular Biology 1987-1988.
J Wiley & Sons, New York
Baker JC,
Orlandi EW
(1995)
Active oxygen in plant pathogenesis.
Annu Rev Phytopathol
33:
299-321
[CrossRef][Web of Science]
Berna A,
Bernier F
(1997)
Regulated expression of a wheat germin gene in tobacco: oxalate oxidase activity and apoplastic localization of the heterologous protein.
Plant Mol Biol
33:
417-429
[CrossRef][Web of Science][Medline]
Bryngelsson T,
Sommer-Knudsen J,
Gregersen PL,
Collinge DB,
Ek B,
Thordal-Christensen H
(1994)
Purification, characterization and molecular cloning of basic PR-1-type pathogenesis-related proteins from barley.
Mol Plant-Microbe Interact
7:
267-275
[Medline]
Collinge DB,
Milligan DE,
Dow JM,
Scofield G,
Daniels MJ
(1987)
Gene expession in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians.
Plant Mol Biol
8:
405-414
[CrossRef]
Devereux J,
Haeberli P,
Smithies O
(1984)
A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res
12:
387-395
Dumas B,
Freyssinet G,
Pallett KE
(1995)
Tissue-specific expression of germin-like oxalate oxidase during development and fungal infection of barley seedlings.
Plant Physiol
107:
1091-1096
[Abstract]
Dumas B,
Sailland A,
Cheviet J-P,
Freyssinet G,
Pallett K
(1993)
Identification of barley oxalate oxidase as a germin-like protein.
CR Acad Sci Paris
316:
793-798
Gregersen PL,
Thordal-Christensen H,
Förster H,
Collinge DB
(1997)
Differential gene transcript accumulation in barley leaf epidermis and mesophyll in response to attack by Blumeria graminis f.sp. hordei.
Physiol Mol Plant Pathol
51:
85-97
[CrossRef]
Harlow E,
Lane D
(1988)
Antibodies: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Hurkman WJ,
Lane BG,
Tanaka CK
(1994)
Nucleotide sequence of a transcript encoding a germin-like protein that is present in salt-stressed barley (Hordeum vulgare L.) roots.
Plant Physiol
104:
803-804
[CrossRef][Web of Science][Medline]
Hurkman WJ,
Tanaka CK
(1996a)
Effect of salt stress on germin expression in barley roots.
Plant Physiol
110:
971-977
[Abstract]
Hurkman WJ,
Tanaka CK
(1996b)
Germin gene expression is induced in wheat leaves by powdery mildew infection.
Plant Physiol
111:
735-739
[Abstract]
Hurkman WJ,
Tao HP,
Tanaka CK
(1991)
Germin-like polypeptides increase in barley roots during salt stress.
Plant Physiol
97:
366-374
[Abstract/Free Full Text]
Knogge W
(1996)
Fungal infection of plants.
Plant Cell
8:
1711-1722
[CrossRef][Web of Science][Medline]
Kølster P,
Munk L,
Stølen O,
Løhde J
(1986)
Near-isogenic barley lines with genes for resistance to powdery mildew.
Crop Sci
26:
903-907
[Abstract/Free Full Text]
Lane BG
(1994)
Oxalate, germin, and the extracellular matrix of higher plants.
FASEB J
8:
294-301
[Abstract]
Lane BG,
Dunwell JM,
Ray JA,
Schmitt MR,
Cuming AC
(1993)
Germin, a protein marker of early plant development, is an oxalate oxidase.
J Biol Chem
268:
12239-12242
[Abstract/Free Full Text]
Levine A,
Pennell RI,
Alvarez ME,
Palmer R,
Lamb C
(1996)
Calcium-mediated apoptosis in a plant hypersensitive disease resistance response.
Curr Biol
6:
427-437
[CrossRef][Web of Science][Medline]
Low PS,
Merida JR
(1996)
The oxidative burst in plant defense: function and signal transduction.
Physiol Plant
96:
533-542
[CrossRef]
Nielsen KK,
Mikkelsen JD,
Kragh KM,
Bojsen K
(1993)
An acidic class III chitinase in sugar beet: induction by Cercospora beticola, characterization and expression in transgenic tobacco plants.
Mol Plant-Microbe Interact
6:
495-506
[Medline]
Price A,
Knight M,
Knight H,
Cuin T,
Tomos D,
Ashenden T
(1996)
Cytosolic calcium and oxidative plant stress.
Biochem Soc Trans
24:
497-483
[Medline]
Pundir CS
(1991)
Purification and properties of oxalate oxidase from Sorghum leaves.
Phytochemistry
30:
1065-1067
[CrossRef]
Sambrook J,
Fritsch EF,
Maniatis T
(1989)
Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Sugiura M,
Yamamura H,
Hirano K,
Sasaki M,
Morikawa M,
Tsuboi M
(1979)
Purification and properties of oxalate oxidase from barley seedlings.
Chem Pharm Bull
27:
2003-2007
Thordal-Christensen H,
Brandt J,
Cho BH,
Rasmussen SK,
Gregersen PL,
Smedegaard-Petersen V,
Collinge DB
(1992)
cDNA cloning and characterization of two barley peroxidase transcripts induced differentially by the powdery mildew fungus Erysiphe graminis.
Physiol Mol Plant Pathol
40:
395-409
[CrossRef]
Thordal-Christensen H,
Smedegaard-Petersen V
(1988)
Comparison of resistance-inducing abilities of virulent and avirulent races of Erysiphe graminis f.sp. hordei and a race of Erysiphe graminis f.sp. tritici in barley.
Plant Pathol
37:
20-27
Thordal-Christensen H,
Zhang Z,
Wei Y,
Collinge DB
(1997)
Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction.
Plant J
11:
1187-1194
[CrossRef][Web of Science]
Trewavas A,
Gilroy S
(1991)
Signal transduction in plant cells.
Trends Genet
7:
356-361
[Medline]
Vera-Estrella R,
Barkla BJ,
Higgins VJ,
Blumwald E
(1994)
Plant defense response to fungal pathogens, activation of host-plasma membrane H+-ATPase by elicitor-induced enzyme dephosphorylation.
Plant Physiol
104:
209-215
[Abstract]
Wei Y,
Zhang Z,
Andersen CH,
Schmelzer E,
Gregersen PL,
Collinge DB,
Smedegaard-Petersen V,
Thordal-Christensen H
(1998)
An epidermis/papilla-specific oxalate oxidase-like protein in the defence response of barley attacked by the powdery mildew fungus.
Plant Mol Biol
36:
101-112
[CrossRef][Web of Science][Medline]
Wevelsiep L,
Rüpping E,
Knogge W
(1993)
Stimulation of barley plasmalemma H+-ATPase by phytotoxic peptides from the fungal pathogen Rhynchosporium secalis.
Plant Physiol
101:
297-301
[Abstract]
Xing T,
Higgins VJ,
Blumwald E
(1996)
Regulation of plant defense response to fungal pathogens: two types of protein kinases in the reversible phosphorylation of the host plasma membrane H+-ATPase.
Plant Cell
8:
555-564
[Abstract]
Zhang Z,
Collinge DB,
Thordal-Christensen H
(1995)
Germin-like oxalate oxidase, a H2O2-producing enzyme, accumulates in barley attacked by the powdery mildew fungus.
Plant J
8:
139-145
Zhang Z,
Yang J,
Collinge DB,
Thordal-Christensen H
(1996)
Ethanol increases sensitivity of oxalate oxidase assay and facilitates direct activity staining in SDS gels.
Plant Mol Biol Rep
14:
266-272
[Web of Science]
This article has been cited by other articles:
|
|
|
|
 
P. M. Manosalva, R. M. Davidson, B. Liu, X. Zhu, S. H. Hulbert, H. Leung, and J. E. Leach
A Germin-Like Protein Gene Family Functions as a Complex Quantitative Trait Locus Conferring Broad-Spectrum Disease Resistance in Rice
Plant Physiology,
January 1, 2009;
149(1):
286 - 296.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. M. Whittaker, H.-Y. Pan, E. T. Yukl, and J. W. Whittaker
Burst Kinetics and Redox Transformations of the Active Site Manganese Ion in Oxalate Oxidase: IMPLICATIONS FOR THE CATALYTIC MECHANISM
J. Biol. Chem.,
March 9, 2007;
282(10):
7011 - 7023.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Zimmermann, H. Baumlein, H.-P. Mock, A. Himmelbach, and P. Schweizer
The Multigene Family Encoding Germin-Like Proteins of Barley. Regulation and Function in Basal Host Resistance
Plant Physiology,
September 1, 2006;
142(1):
181 - 192.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Prats, A. P. Gay, L. A. J. Mur, B. J. Thomas, and T. L. W. Carver
Stomatal lock-open, a consequence of epidermal cell death, follows transient suppression of stomatal opening in barley attacked by Blumeria graminis
J. Exp. Bot.,
July 1, 2006;
57(10):
2211 - 2226.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Opaleye, R.-S. Rose, M. M. Whittaker, E.-J. Woo, J. W. Whittaker, and R. W. Pickersgill
Structural and Spectroscopic Studies Shed Light on the Mechanism of Oxalate Oxidase
J. Biol. Chem.,
March 10, 2006;
281(10):
6428 - 6433.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. R. Escutia, L. Bowater, A. Edwards, A. R. Bottrill, M. R. Burrell, R. Polanco, R. Vicuna, and S. Bornemann
Cloning and Sequencing of Two Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Allelic Isoforms: Implications for the Reaction Specificity of Oxalate Oxidases and Decarboxylases
Appl. Envir. Microbiol.,
July 1, 2005;
71(7):
3608 - 3616.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. M. Livingstone, J. L. Hampton, P. M. Phipps, and E. A. Grabau
Enhancing Resistance to Sclerotinia minor in Peanut by Expressing a Barley Oxalate Oxidase Gene
Plant Physiology,
April 1, 2005;
137(4):
1354 - 1362.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Hu, D. L. Bidney, N. Yalpani, J. P. Duvick, O. Crasta, O. Folkerts, and G. Lu
Overexpression of a Gene Encoding Hydrogen Peroxide-Generating Oxalate Oxidase Evokes Defense Responses in Sunflower
Plant Physiology,
September 1, 2003;
133(1):
170 - 181.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Yoshioka, N. Numata, K. Nakajima, S. Katou, K. Kawakita, O. Rowland, J. D. G. Jones, and N. Doke
Nicotiana benthamiana gp91phox Homologs NbrbohA and NbrbohB Participate in H2O2 Accumulation and Resistance to Phytophthora infestans
PLANT CELL,
March 1, 2003;
15(3):
706 - 718.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Talarczyk, M. Krzymowska, W. Borucki, and J. Hennig
Effect of Yeast CTA1 Gene Expression on Response of Tobacco Plants to Tobacco Mosaic Virus Infection
Plant Physiology,
July 1, 2002;
129(3):
1032 - 1044.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Torres, J. L. Dangl, and J. D. G. Jones
Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response
PNAS,
December 21, 2001;
(2001)
12452499.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. S. Dahleen, P. A. Okubara, and A. E. Blechl
Transgenic Approaches to Combat Fusarium Head Blight in Wheat and Barley
Crop Sci.,
May 1, 2001;
41(3):
628 - 637.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. N. Dodds, G. J. Lawrence, and J. G. Ellis
Six Amino Acid Changes Confined to the Leucine-Rich Repeat {beta}-Strand/{beta}-Turn Motif Determine the Difference between the P and P2 Rust Resistance Specificities in Flax
PLANT CELL,
January 1, 2001;
13(1):
163 - 178.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
S. G. Cessna, V. E. Sears, M. B. Dickman, and P. S. Low
Oxalic Acid, a Pathogenicity Factor for Sclerotinia sclerotiorum, Suppresses the Oxidative Burst of the Host Plant
PLANT CELL,
November 1, 2000;
12(11):
2191 - 2200.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
J. M. Dunwell, S. Khuri, and P. J. Gane
Microbial Relatives of the Seed Storage Proteins of Higher Plants: Conservation of Structure and Diversification of Function during Evolution of the Cupin Superfamily
Microbiol. Mol. Biol. Rev.,
March 1, 2000;
64(1):
153 - 179.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Hückelhoven, J. Fodor, C. Preis, and K.-H. Kogel
Hypersensitive Cell Death and Papilla Formation in Barley Attacked by the Powdery Mildew Fungus Are Associated with Hydrogen Peroxide but Not with Salicylic Acid Accumulation
Plant Physiology,
April 1, 1999;
119(4):
1251 - 1260.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
M. A. Torres, J. L. Dangl, and J. D. G. Jones
Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response
PNAS,
January 8, 2002;
99(1):
517 - 522.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Druka, D. Kudrna, C. G. Kannangara, D. von Wettstein, and A. Kleinhofs
Physical and genetic mapping of barley (Hordeum vulgare) germin-like cDNAs
PNAS,
January 22, 2002;
99(2):
850 - 855.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction