MPP_126.
fm Page 329 Wednesday, August 21, 2002 6:38 PM
MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345
Blackwell Science, Ltd
Primary structure and tissue-specific expression of
the pathogenesis-related protein PR-1b in potato†
E R I K A H O E G E N 1 , ‡ , A N I T A S T R Ö M B E R G 2 , U L L A P I H L G R E N 2 A N D E R I C H KO M B R I N K 1,*
1
Max-Planck-Institut für Züchtungsforschung, Abteilung Biochemie, Carl-von-Linné-Weg 10, 50829 Köln, Germany
2
Uppsala Genetic Center, Department of Plant Biology, Swedish University of Agricultural Sciences, Box 7055, S-750 07 Uppsala, Sweden
SUMMARY
The infection of potato (Solanum tuberosum) leaves with the late
blight pathogen Phytophthora infestans , or treatment with
fungal elicitor, leads to the massive accumulation of pathogenesis-
related (PR) proteins in the extracellular leaf space. The most
abundant of these proteins was purified to apparent homogene-
ity and identified as a new, basic member of the PR-1 family of
defence proteins, designated PR-1b. Antibodies raised against
the protein and a cDNA isolated by differential screening were
used to study the temporal and spatial patterns of PR-1b protein
and mRNA distribution in healthy and infected potato tissues. PR-
1b was present in old leaves and at low levels also in the carpels
of flowers. In leaves, strong accumulation of PR-1b mRNA and
protein occurred in response to infection by the oomycete pathogen
Phytophthora infestans or the bacterial pathogen Pseudomonas
syringae pv. maculicola. PR-1b mRNA and protein accumula-
tion was clearly initiated at the infection site, but a delayed
and sustained accumulation was also observed in neighbouring,
uninfected leaves of potato plants. Tissue- and cell type-specific
expression of PR-1b was analysed by immunohistochemical
and in situ RNA hybridization techniques. Appreciable amounts
of PR-1b protein and mRNA were localized in epidermal cells,
guard cells of the stomata, glandular trichomes, crystal idioblasts,
and cells of the vascular system of infected leaves. However, no
significant differences in the amounts and distribution patterns of
PR-1b could be observed between compatible and incompatible
interactions of potato and Phytophthora infestans, indicating
that PR-1b expression is not involved in determining cultivar /
race-specific resistance in potato.
*Correspondence : E-mail: kombrink@mpiz-koeln.mpg.de
†Database accession: the sequence of the PR-1b precursor reported in this paper has been
deposited at GENBANK with the accession number AY050221.
‡Present address : AMAXA GmbH, Gottfried-Hagen-Str. 60-62, 51105 Köln, Germany.
© 2002 BLACKWELL SCIENCE LTD
I N T RO D U C T I O N
Plants have evolved a large variety of sophisticated defence
mechanisms to defend themselves against microbial infections.
Preformed physical barriers, such as the plant cell wall, and
antimicrobial compounds constitute the first line of defence
(Kombrink and Somssich, 1995; Osbourn, 1996). Superimposed
upon these are the active defence mechanisms, which are engaged
in response to attempted microbial ingress (Hammond-Kosack
and Jones, 1996; Kombrink and Somssich, 1995). A key feature
of active pathogen defence is the rapid induction of host gene
expression, which leads to the de novo synthesis of numerous
specific proteins (Kombrink and Somssich, 1995). The most abund-
ant and probably best characterized of these are the pathogenesis-
related (PR) proteins, which comprise several structurally and
functionally distinct families that are induced in a large variety
of plant species (van Loon, 1999; van Loon and van Strien, 1999).
PR proteins accumulate coordinately in host plants not only
after pathogen infection, but also after abiotic stresses, such as
wounding or treatment with elicitors or chemical inducers, e.g.
salicylic acid, ethylene, and jasmonic acid; this suggests a general
function of these proteins in adaptation to biotic stress conditions
and pathogen defence in particular (Kombrink and Somssich,
1997; van Loon and van Strien, 1999).
Currently, 14 distinct families of PR proteins are known in
plants (van Loon and van Strien, 1999). Some families are
composed of proteins with identified biochemical function, e.g.
1,3-β-glucanases (PR-2), chitinases (PR-3, PR-4, PR-8, PR-11),
proteinase inhibitors (PR-6), or peroxidases (PR-9), which support
their participation in pathogen defence (Kombrink and Somssich,
1997; van Loon and van Strien, 1999). However, for other PR
proteins, including PR-1, the functions remain enigmatic. Most PR
proteins occur in both basic and acidic isoforms, which differ in
their expression and cellular localization (Brederode et al., 1991;
Memelink et al., 1990). In tobacco, the genes encoding acidic PR
proteins are induced to high levels not only locally in close vicinity
to the invading pathogen, but also in distant, uninfected parts of
the plant (Kombrink and Somssich, 1997; van Loon and van Strien,
1999). This systemic induction is closely correlated with the
329
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330 E. HOEGEN et al.
accumulation of the signal molecule salicylic acid and the phe-
nomenon of systemic acquired resistance (SAR), a defence mech-
anism that establishes durable, broad-spectrum pathogen resistance
(Ryals et al., 1996; Sticher et al., 1997). The acidic PR proteins in
tobacco are predominantly secreted into the plant apoplast and
accumulate extracellularly, whereas their basic counterparts are
retained in the cell and deposited in the vacuole (Kombrink and
Somssich, 1997). Basic PR proteins are usually not expressed sys-
temically and are not induced by salicylic acid treatment. In contrast,
their expression is strongly stimulated by the plant hormone ethylene
(Brederode et al., 1991; Eyal et al., 1992; Memelink et al., 1990),
which is produced in most plant tissues in response to infection.
The most abundant PR proteins, at least in tobacco, are those
found in family PR-1. They also occur in acidic and basic isoforms
and the former are induced up to 10 000-fold in infected tissue,
reaching levels of up to 2% of the total leaf protein (Alexander
et al., 1993; Buchel and Linthorst, 1999). PR-1-like proteins occur
not only in plants, but are also present in other eukaryotic organ-
isms, such as fungi, yeasts, insects, and vertebrates, including
humans, suggesting that the encoding genes arose relatively
early during evolution, which is indicative of a fundamental and
ancient cellular function (Buchel and Linthorst, 1999; van Loon
and van Strien, 1999). However, their function in these other
organisms is likewise not known. The first evidence, albeit indir-
ect, for the participation of PR-1 in plant defence was obtained
with transgenic tobacco plants, expressing the PR-1a gene at
high levels. These plants exhibited increased tolerance to the
oomycete pathogens Phytophthora parasitica var. nicotianae and
Peronospora tabacina (Alexander et al., 1993). Direct antifungal
activity of various purified tobacco and tomato PR-1 proteins
against Phytophthora infestans was more recently demonstrated
both in vitro, by the inhibition of zoospore germination, and in
vivo, by the reduced colonization of leaf discs (Niderman et al.,
1995). In these experiments, the basic PR-1 proteins PR-1g
(tobacco) and P14c (tomato), which showed considerable sequence
similarity, were the most effective, whereas the acidic PR-1 pro-
teins (tobacco) were only slightly inhibitory.
In tobacco, the acidic PR-1 isoforms are predominant and
are localized in the extracellular spaces and xylem elements of
tobacco mosaic virus (TMV)-infected leaves (Buchel and Linthorst,
1999). Their pronounced stability and resistance to proteolytic
degradation and acidic pH conditions make them highly adaptive
to the conditions of the extracellular environment. In contrast,
the basic PR-1 proteins of tobacco are retained in the cell and
accumulate in the vacuole (Buchel and Linthorst, 1999; Eyal
et al., 1993; Sessa et al., 1995). The presence of carboxy-terminal
extensions in these proteins is believed to be important for
vacuolar targeting (Melchers et al., 1993; Neuhaus et al., 1991). In
other plants, extracellular proteins corresponding to the acidic
PR-1 proteins of tobacco do not necessarily have low isoelectric
points. In tomato, the most abundant proteins purified from the
MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345 © 2002 BLACKWELL SCIENCE LTD
apoplastic fluid of infected leaves are the basic PR-1 isoforms P4
and P6 (P14) (Joosten et al., 1990; van Kan et al., 1992), whereas
the acidic isoforms carry C-terminal extensions for vacuolar
targeting (Tornero et al., 1994, 1997).
We have previously purified various PR proteins and cloned
their respective genes from potato, in particular 1,3-β-glucanases
and chitinases, and have exploited the race/cultivar-specific
interaction between potato and the late blight pathogen Phyto-
phthora infestans to analyse in detail their temporal and spatial
expression patterns by immunohistochemistry and in situ RNA
hybridization techniques (Ancillo et al., 1999; Beerhues and
Kombrink, 1994; Büchter et al., 1997; Kombrink et al., 1988,
1993; Schröder et al., 1992; Taylor et al., 1990). In view of the
accumulating evidence that proteins of the PR-1 type may be
essential defence components against oomycete pathogens, we
extended these investigations to the most abundant PR proteins
present in the apoplastic space of infected potato leaves. Here,
we report the purification and molecular analysis of a new, basic
PR-1 protein from potato. The possible functional implications of
the organ- and cell type-specific protein and mRNA distribution
for plant defence will be discussed.
RESULTS
Accumulation of PR proteins
Inoculation of potato leaves with Phytophthora infestans led to
the massive accumulation of PR proteins. The total amount of
protein extractable with the intercellular washing fluid (IWF)
increased on infection from about 0.1 to 1 mg/mL, and analysis
by sodium dodecylsulphate polyacrylamide gel electrophoresis
(SDS-PAGE) revealed that a drastic alteration of the protein
pattern occurred (Fig. 1). While the relative abundance of proteins
with high molecular weight decreased, several proteins in the
molecular weight range 10 000– 40 000 increased in concentra-
tion. When compatible and incompatible interactions were
compared, no significant differences in the accumulation of these
PR proteins could be observed up to 84 h post-inoculation (Fig. 1).
However, at later infection stages, the protein levels remained
high in the incompatible interaction (Fig. 1A), whereas they
decreased in the compatible interaction (Fig. 1B). Similar differ-
ential expression patterns, i.e. higher induction in the compatible
compared to the incompatible interaction, have been reported for
other activated defence responses in infected potato leaves, such
as the accumulation of phenylalanine ammonia-lyase (PAL), 4-
coumarate:coenzyme A ligase (4CL), glutathione S-transferase (GST),
1,3-β-glucanase, and chitinase mRNAs or proteins (Becker-André
et al., 1991; Fritzemeier et al., 1987; Hahn and Strittmatter, 1994;
Schröder et al., 1992; Taylor et al., 1990).
The IWF proteins with Mr = 28 000 – 36 000 were previously
identified as 1,3-β-glucanases and chitinases (Ancillo et al., 1999;
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Pathogenesis-related protein PR-1b from potato 331

Fig. 1 Accumulation of pathogenesis-related (PR)

proteins in potato leaves infected with Phytophthora
infestans. Leaves of potato cultivar Datura were
inoculated by spraying with a spore suspension
(105 spores/mL) of P. infestans race 4 (A) or race 1
(B), resulting in an incompatible or compatible
interaction, respectively. At the times indicated, the
intercellular washing fluid was isolated and, after
concentration, equal amounts of protein (150 µg/
lane) were separated by sodium dodecylsulphate
polyacrylamide gel electrophoresis (SDS-PAGE) and
stained with Coomassie Brilliant Blue. During the
infection process, the protein content of the
intercellular washing fluid increased from 100 µg/
mL (0 h) to 800 µg/mL (136 h). The positions of
previously purified 1,3-β-glucanases (GluB) and
chitinases (ChtA) are marked by arrowheads.

Kombrink et al., 1988). In contrast, the most abundant protein
found in the IWF (Mr = 15 000) has not yet been characterized
from potato, although its size and extracellular localization sug-
gest that it could be a PR-1-type protein. We purified the protein
to apparent homogeneity from the IWF of elicitor-treated potato
leaves by ion exchange fast protein liquid chromatography (FPLC)
and preparative SDS-PAGE as described in the ‘Experimental
procedures’ section. In fact, the protein was resolved into two
isoforms with identical molecular weight ( Mr = 15 000) and
slightly different charge (pI = 9.7 and 9.0; Fig. 2), and the isoform
with basic pI was tentatively designated as PR-1b.
Antisera raised in rabbits against purified PR-1b were used
to study the biosynthesis and serological relationship of potato PR
proteins, as well as their cellular localization. When applied to protein
blots containing IWF of elicitor-treated potato leaves, the antiserum
recognized a single protein band of identical size to the purified
protein (Fig. 3A). No immunological cross-reaction to any other
protein was observed. In extracts of leaves from which the extra-
cellular proteins had been removed with the IWF, the 15-kDa
protein was also detectable but, in addition, a second protein
(Mr = 16 000) of lower abundance cross-reacted with the antiserum
(Fig. 3B). Whether this represents the intracellular precursor of the
secreted PR-1b protein or another, unrelated protein is at present
unknown. Elicitor treatment resulted in a rapid and massive accu-
mulation of PR-1b which, in untreated leaves, was barely detectable.
A virtually identical pattern of PR-1b accumulation was observed
in potato leaves after inoculation with Phytophthora infestans
(not shown). Low amounts of PR-1b were also detectable in old
leaves, roots, and tubers of non-infected potato plants, whereas
it was absent from young leaves and stems (not shown).
Isolation and analysis of PR-1b cDNA
Differential screening for pathogen-inducible potato genes had
previously resulted in the isolation of a small cDNA fragment
(200 bp in length) with high sequence similarity to the protein
P14 from tomato (Hoegen, 1998; Tornero et al., 1993; van Kan
et al., 1992). When this fragment was used for screening of a
cDNA library, representing the mRNA of systemically activated
potato leaves, two near full-length cDNA clones were isolated
that were identical in sequence but differed in length. The longer
cDNA clone (693 bp) was used for all further experiments
(GENBANK accession number AY050221). It contains an open reading
frame of 480 bp, encoding a protein of 159 amino acids. At the
N-terminus of the deduced amino acid sequence, a putative
hydrophobic signal peptide of 24 amino acids can be identified
(Fig. 4). Removal of this peptide according to the rules of von
Heijne (von Heijne, 1983) results in a mature protein with a cal-
culated Mr of 14 729 and pI of 8.22. The unprocessed protein has
a calculated Mr of 17 337 and a pI of 7.8. These properties are in
good agreement with those determined experimentally for the
purified PR-1b protein (see above).

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332 E. HOEGEN et al.
Fig. 2 Properties of PR-1b purified to apparent homogeneity from potato leaves.
Proteins eluting in two separate peaks at 90 mM NaCl (lane 1) and 110 mM NaCl
(lane 2) from the final cation exchange fast protein liquid chromatography (FPLC)
column were separated on sodium dodecylsulphate polyacrylamide (A) and
isoelectric focusing (B) gels. Proteins (13 µg/lane in A, 6 µg / lane in B) were stained
with Coomassie Brilliant Blue. Lane 3, Mr (A) and pI (B) marker proteins.
Database searches with the predicted PR-1b amino acid sequence
revealed high sequence similarity to PR-1 proteins from several
plants. The sequence alignment further uncovered a character-
istic feature of all PR-1 proteins, namely the presence of six strictly
MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345 © 2002 BLACKWELL SCIENCE LTD
conserved cysteine residues (Fig. 4). Two basic PR-1 isoforms
from tomato, designated PR-1b1 and PR-1b2, also described as P6
(P14) and P4, respectively (Tornero et al., 1994; van Kan et al.,
1992), showed the greatest similarity (91% identity at the amino
acid level) to potato PR-1b. These proteins also accumulate in
the extracellular leaf space after infection (Joosten et al., 1990;
Tornero et al., 1994). In contrast, the secreted PR-1 isoforms of
tobacco, PR-1a, PR-1b, and PR-1c (Cutt et al., 1988), are acidic in
nature and exhibit only 60% identity to potato PR-1b. The same
level of sequence similarity is also shared with other acidic PR-1
proteins, for example those from Arabidopsis thaliana and Brassica
napus. All these PR proteins are secreted because they lack a
retention signal for vacuolar targeting, which is a short peptide
extension located at the C-termini of the proteins (Melchers
et al., 1993; Neuhaus et al., 1991). Such retention signals are
present in the basic PR-1 proteins of tobacco, designated PRB-1b
(Eyal et al., 1992), and bell pepper (Capsicum annuum), as well
as the acidic PR-1a1 protein from tomato, and hence these
proteins accumulate in the vacuole (Sessa et al., 1995; Tornero et al.,
1994). With these intracellular PR-1 isoforms, the potato PR-1b
protein shared sequence identities of 66%, 62% and 55%,
respectively.
To prove that the isolated clones encode the purified PR-1b
protein, the complete cDNA, including the N-terminal signal
sequence, was cloned into the bacterial expression vector pQE51
behind an isopropyl-1-thio- β-D-galactoside (IPTG)-inducible
promoter and expressed in Escherichia coli. Bacterial extracts
were separated by SDS-PAGE, blotted on to nitrocellulose, and
reacted with PR-1b-specific antiserum. The results shown in Fig. 5
demonstrate that, in E. coli, PR-1b is presumably localized in inclu-
sion bodies, as only relatively low amounts were detectable in
the soluble fraction, whereas the SDS-treated, insoluble fraction
contained high amounts of a protein with an apparent Mr of
18 000, corresponding to unprocessed PR-1b. In addition to the
Fig. 3 Immunoblot analysis of PR-1b induction in
potato leaves. Detached leaves of potato (cv. Datura)
were treated with fungal elicitor (100 µg/mL) or
water (control) for the times indicated. Extracellular
proteins were extracted with the intercellular
washing fluid (IWF) and cellular proteins were
obtained from the remainder of the leaves. Proteins
(100 µL for IWF, 15 µg for cell extract) were
separated by sodium dodecylsulphate
polyacrylamide gel electrophoresis (SDS-PAGE),
transferred to nitrocellulose, and probed with
antiserum raised against purified PR-1b at a dilution
of 1 : 5000 for 2 h.
MPP_126.fm Page 333 Wednesday, August 21, 2002 6:38 PM

Pathogenesis-related protein PR-1b from potato 333

Fig. 4 Predicted amino acid sequence of the potato

PR-1b protein and comparison with similar PR-1
proteins from other plants. Protein sequences were
aligned with the program PILEUP and grouped
according to their similarity to potato PR-1b. Gaps (.)
were introduced for optimal alignment. Amino acids
common to at least six of the eight sequences
are shaded. The putative cleavage site of the
hydrophobic signal peptide, which is present at the
N-terminus of all proteins, is marked by an arrow.
The six cysteine residues that are strictly conserved in
all plant PR-1 proteins are denoted by asterisks.
Abbreviations for the proteins presented in the
figure are as follows (with SWISS-PROT/ GENBANK
database accession numbers given in parentheses):
St, Solanum tuberosum PR-1b (AAL01594);
Le, Lycopersicon esculentum PR-1b1 (P04284);
Nt, Nicotiana tabacum basic PRB-1b (Q04106); Le,
Lycopersicon esculentum PR-1a1 (Q08697); Ca,
Capsicum annuum PR-1 (O65157); Nt, Nicotiana
tabacum acid PR-1a (P08299); At, Arabidopsis
thaliana PR-1 (P33154); Bn, Brassica napus PR-1
(Q43392).

major band, the antiserum cross-reacted with a second protein
(Mr = 15 000), which presumably represents a processed or
degraded form of PR-1b. In extracts of E. coli cells containing
the empty expression vector, no proteins cross-reacting with the
PR-1b antiserum could be detected. From these results, we con-
clude that the isolated cDNA encodes one of the PR-1b isoforms
expressed in potato leaves.
Expression of PR-1b mRNA in potato plants
In several plants, it has been demonstrated that PR-1 proteins
accumulate in response to infection, elicitor treatment, and other
kinds of stimuli as a result of transcriptional activation of the
corresponding genes. Infection of potato leaves by Phytophthora
infestans caused a strong increase in PR-1b mRNA amounts,
starting at 18 h post-inoculation and reaching maximum steady
state levels at 36 h (Fig. 6). Thus, the transient accumulation of
mRNA precedes that of the protein by several hours (cf. Fig. 1)
and closely resembles the induction pattern of other PR proteins,
such as acidic (class II) chitinase, ChtA (Fig. 6). A similar time
course of induction is displayed by basic (class I) chitinase and
1,3-β-glucanase (Beerhues and Kombrink, 1994). In contrast,
other activated defence responses, such as cell wall appositions,
hypersensitive cell death, or the expression of genes encoding
enzymes of the general phenylpropanoid pathway (PAL, 4CL),
were induced at a much faster rate, with the respective mRNA
amounts being detectable as early as 3 – 6 h post-inoculation
(Freytag et al., 1994; Fritzemeier et al., 1987). When potato leaves
were inoculated with the bacterial pathogen Pseudomonas
syringae pv. maculicola, the time course of PR-1b mRNA accu-
mulation was similar to that observed after inoculation with
Phytophthora infestans (Fig. 6B).
Wounding or ethylene treatment of potato leaves also resulted
in a moderate accumulation of PR-1b mRNA at relatively late
time points after treatment (72 h), whereas treatment with
salicylic acid or jasmonic acid did not cause any detectable induc-
tion (results not shown).
When different organs and developmental stages of potato
plants were analysed by RNA blot analysis, high amounts of PR-
1b mRNA were detectable in old leaves; lower amounts were

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334 E. HOEGEN et al.
present in leaves of intermediate age and in carpels (Fig. 6C). All
other analysed organs, including young leaves, stems, roots and
tubers, were devoid of detectable amounts of PR-1b mRNA. Thus,
PR-1b expression is apparently not only controlled by infection,
but is also dependent on the developmental stage of the leaf. This
is in accordance with our previous finding that uninfected potato
leaves contain only low levels of PR-1b protein (Fig. 1). Similar
developmental- and organ-specific expression patterns have also
been reported for other PR proteins in potato and other plants
(Ancillo et al., 1999; Beerhues and Kombrink, 1994; Buchel and
Linthorst, 1999; Lotan et al., 1989; van Loon, 1999).
Immunohistochemical localization of PR-1b in infected
potato leaves
The monospecific antiserum against PR-1b allowed us to obtain
more precise information on the cellular localization of the
protein and its accumulation in relation to pathogen growth in
infected plants. The results of a detailed analysis of numerous
infected potato leaves with compatible or incompatible inter-
action (potato cv. Datura inoculated with Phytophthora infestans
race 1 or 4, respectively) are presented in Figs 7 and 8. Strong PR-
1b accumulation in the vicinity of the successfully colonized leaf
area is evident from the intense brown labelling of the whole
tissue section and of the epidermal cell layer in particular (Fig. 7A).
The infection sites were easily identified by the collapsed
MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345 © 2002 BLACKWELL SCIENCE LTD
Fig. 5 Analysis of heterologously expressed PR-1b
by sodium dodecylsulphate (SDS) polyacrylamide
gels. Escherichia coli cells harbouring the pQE51
expression vector with the PR-1b cDNA (lanes 1 and
3) or the empty control vector (lanes 2 and 4) were
treated with isopropyl-1-thio-β-D-galactoside (IPTG)
to induce the expression of the heterologous
protein. Cellular extracts obtained in the absence
(lanes 1 and 2) or presence (lanes 3 and 4) of SDS
were separated by SDS-PAGE. Proteins were stained
with Coomassie Brilliant Blue (A) or blotted on to
nitrocellulose and probed with an antiserum raised
against purified PR-1b at a dilution of 1 : 2000 for
2 h (B).
morphology of the penetrated epidermal cells, and pathogen biomass
was detectable by a Phytophthora-specific antiserum, resulting in
brown staining of the leaf area colonized by the pathogen (Fig. 7B).
In tissue sections of the same leaf, but taken at a distance from
the infection site proper, the preferential accumulation of PR-1b
in the epidermis in comparison to the leaf mesophyll is retained
and, in addition, strong labelling in guard cells of the stomata can
be detected (Fig. 7C). In addition to a general labelling of the cell
cytoplasm, intense staining of the apoplast revealed that PR-1b
was preferentially localized in the intercellular and subcuticular leaf
spaces (Fig. 7E), which is in accordance with its easy extraction with
the IWF. The absence of any labelling in parallel sections incubated
with Phytophthora-specific antiserum or preimmune serum as
control demonstrated the specificity of PR-1b detection (Fig. 7D,F).
An additional location within infected leaves containing high
amounts of PR-1b involved specialized cells known as crystal
idioblasts (Fig. 7G,I). Crystal idioblasts differentiate from paren-
chyma cells and have a distinct morphology due to the presence
of calcium oxalate crystals within their vacuoles. They could be
readily identified in leaf sections due to their distinctive granular
morphology and their occurrence within the leaf between the
upper palisade layer and the lower spongy mesophyll (Fig. 7G–J).
PR-1b accumulated not only within crystal idioblasts that were
located close to infection sites (Fig. 7G), but also within those of
distal tissue sections (Fig. 7I). However, PR-1b was not detected
within crystal idioblasts of non-infected potato leaves. No labelling
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Pathogenesis-related protein PR-1b from potato 335

Fig. 6 RNA blot analysis of PR-1b expression in

different potato organs. Detached potato leaves (cv.
Datura) were infected with Phytophthora infestans
(race 1) (A) or Pseudomonas syringae pv. maculicola
(B) and incubated for the times indicated. Control
leaves were treated with water or MgCl2 solution
and maintained in parallel under otherwise identical
conditions. For organ- and development-specific
expression (C), leaves of a 6 – 8-week-old plant were
divided into four groups of three consecutive leaves
each: 1, youngest leaves (top of the plant); 2 and 3,
intermediate stages; 4 oldest leaves (bottom of the
plant). Stems were divided into two samples: 1, top
of the plant (0–2 cm); 2, remainder of the plant (2–
50 cm). Fully developed flowers were separated into
petals (P), stamens (St), pedicel (Pe), sepals (S), and
carpels (C). Total extractable RNA (10 µg/lane) was
separated, blotted, and hybridized with
radiolabelled PR-1b cDNA. Equal loading of RNA
was monitored by ethidium bromide staining.

of crystal idioblasts was observed with antisera directed against
potato 1,3-β-glucanase (PR-2) or chitinase (PR-3), demonstrating
the selective restriction of only PR-1b to crystal idioblasts (results
not shown).
Specific labelling with PR-1b-specific antiserum of other tissues
and cell types, such as xylem (Fig. 8A), tracheids (Fig. 8C), and
glandular trichomes (Fig. 8E), was also frequently observed in
tissue sections derived from infected leaves. However, when com-
paring compatible (Datura/Pi1; Fig. 7) and incompatible (Datura /
Pi4; Fig. 8) interactions between potato and Phytophthora
infestans, no significant or distinct differences in the labelling
patterns, and hence cell type-specific localization of PR-1b, could
be detected. A preferential accumulation of PR-1b in the epider-
mis, crystal idioblasts, glandular trichomes, and guard cells of the
stomata was observed in both cases (Fig. 8G–J).
In situ localization of PR-1b mRNA
The cell type-specific expression of PR-1b was also studied
by in situ mRNA hybridization. In these experiments, Histowax-
embedded serial sections of infected and non-infected potato
leaves were hybridized with digoxigenin-labelled antisense and
sense (control) RNA probes of PR-1b. The presence of PR-1b mRNA
was revealed by the formation of a purple/blue precipitate from the

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336 E. HOEGEN et al.
chromogenic substrates and peroxidase-labelled antidigoxigenin.
Infection sites were identified by staining parallel sections for
Phytophthora infestans and by the collapsed morphology of the
penetrated cells (Fig. 9A–C).
In the infected leaves, enhanced tissue labelling was observed
in the vicinity of infection sites when compared to control
MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345 © 2002 BLACKWELL SCIENCE LTD
Fig. 7 Immunolocalization of PR-1b in cross-
sections of potato leaves (cv. Datura) of a compatible
interaction with Phytophthora infestans (race 1).
Leaves were inoculated with a spore suspension
(5 × 105 spores/mL) and harvested at various
times thereafter: A and B, 54 h; C and D, 48 h;
E and F, 42 h; G and H, 60 h; I and J, 72 h. After
fixation, parallel tissue sections were incubated
with antiserum raised against PR-1b (A, C, E, G, I),
antiserum raised against Phytophthora sp. (B) for
detection of pathogen biomass within the tissue, or
preimmune serum (D, F, H, J) followed by peroxidase-
conjugated secondary antibodies against rabbit
IgG. Infection sites are marked by arrowheads;
ci, crystal idioblast; e, epidermis; gc, guard cells;
is, intercellular space; tr, tracheid.
sections (Fig. 9A–C). Specific cell types accumulating PR-1b mRNA
included epidermal cells, guard cells of the stomata, and glandu-
lar trichomes (Fig. 9A,C), whereas labelling of the xylem, although
consistently observed in our experiments, was most probably an
artefact as this tissue consists of non-living (dead) cells without
active metabolism and functional mRNAs.
MPP_126.fm Page 337 Wednesday, August 21, 2002 6:38 PM

Pathogenesis-related protein PR-1b from potato 337

Fig. 8 Immunolocalization of PR-1b in

cross-sections of potato leaves (cv. Datura) of an
incompatible interaction with Phytophthora
infestans (race 4). Leaves were inoculated with a
spore suspension (5 × 105 spores/mL) and harvested
at various times thereafter: A and B, 48 h; C and D,
60 h; E and F, 30 h; G and H, 54 h; I and J, 60 h. After
fixation, parallel tissue sections were incubated with
antiserum raised against PR-1b (A, C, E, G, I) or
preimmune serum (B, D, F, H, J) followed by
peroxidase-conjugated secondary antibodies
against rabbit IgG. Abbreviations: ci, crystal
idioblast; e, epidermis; gc, guard cells; gt, glandular
trichome; tr, tracheid; x, xylem.

A longitudinal section through an infected leaf confirmed the
pronounced cell type-specific expression of PR-1b mRNA in
epidermal cells and guard cells of the stomata (Fig. 9D). Weak
labelling of the leaf vein was also consistently observed (Fig. 9D).
In cross-sections of the same infected leaf, but taken at a distance
from the infection site, the preferential accumulation of PR-1b
mRNA in guard cells, glandular trichomes, and the vascular
system was retained, whereas the expression in the epidermis was
weaker or even absent (Fig. 9F,H). In leaves of uninfected plants,
similar expression patterns were observed, but the overall expres-
sion levels were lower (results not shown). In cross-sections
through the mid-vein of infected potato leaves, labelling of both
the internal and external bundles of the bicollateral phloem
was clearly detectable, as well as labelling of the cytoplasm of
collenchyma cells (Fig. 9J). When comparing locally and systemic-
ally induced expression, no significant differences in the corres-
ponding patterns were observed, with the exception that total
mRNA amounts were higher in directly infected leaves.

© 2002 BLACKWELL SCIENCE LTD MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345
MPP_126.fm Page 338 Wednesday, August 21, 2002 6:38 PM
338 E. HOEGEN et al.
The expression of PR-1b mRNA in crystal idioblasts, as previ-
ously reported by Dixon et al. (1991), could not be observed with
confidence during our experiments. However, during the extens-
ive manipulation of tissue sections, as required for in situ mRNA
localization, preferential damage of these cells due to their high
MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345 © 2002 BLACKWELL SCIENCE LTD
Fig. 9 In situ localization of PR-1b mRNA in
sections of potato leaves. Two leaves of a potato
plant (cv. Désirée) were inoculated with spore
suspension (5 × 105 spores/mL) of Phytophthora
infestans (race 1) resulting in a compatible
interaction. After 45 h, infected leaves (A–E) and
non-inoculated leaves of the same plant (F–K) were
harvested, fixed, and parallel sections hybridized
with antisense (A, C, D, F, H, J) or sense (B, E, G, I, K)
RNA derived from PR-1b cDNA. D and E,
longitudinal leaf sections; all other micrographs
show cross-sections. A positive PR-1b signal is
visualized by the purple/blue precipitate.
Arrowheads mark infection sites. Abbreviations:
c, collenchyma; e, epidermis; ep, external phloem;
gc, guard cells; gt, glandular trichome; ip, internal
phloem; lv, leaf vein; x, xylem.
content of calcium oxalate crystals, and a concomitant loss of
their cellular constituents, may have occurred. The absence of
any labelling in parallel sections with sense RNA probes as control
clearly demonstrated the specificity of PR-1b mRNA detection
(Fig. 9B,E,G,I,K).
MPP_126.fm Page 339 Wednesday, August 21, 2002 6:38 PM
DISCUSSION
Oomycetes represent some of the economically most important
plant pathogens, including the vast range of Peronospora,
Pythium, and Phytophthora species, which cause considerable
losses in crop yield world-wide. Well-known examples are
Phytophthora infestans, the causal agent of the potato late blight
disease, Phytophthora parasitica, causing the tobacco black
shank disease, and downy mildews, such as Peronospora tabacina,
causing the tobacco blue mould disease (Govers et al., 1997).
Control of these extremely destructive pathogens at present
relies mainly on the intensive application of fungicides. To change
from this practice to an increased exploitation of natural resist-
ance mechanisms, an improved knowledge of the underlying
molecular control mechanisms and their histological and cytolo-
gical consequences is required.
Towards this goal, we purified and cloned the most abundant
extracellular protein from potato leaves and identified it as a
member of the PR-1 family of plant defence proteins. The potato
protein, designated PR-1b, exhibited the highest sequence
similarity to basic PR-1 isoforms of other solanaceous plants, in
particular the extracellular PR-1b1 from tomato (Tornero et al.,
1994) and the intracellular PRB-1b from tobacco (Eyal et al.,
1992), whereas the similarity to acidic isoforms was considerably
lower. It is interesting to note that the extra- and intracellular
PR-1 isoforms in tomato and potato have inverse properties in
relation to those expressed in tobacco. Nevertheless, the cellular
localization is consistent with the structural features of the pro-
teins, and retention within the cell is dependent on the presence
of a C-terminal extension that functions as a vacuolar targeting
signal (Melchers et al., 1993; Neuhaus et al., 1991). An intra-
cellular PR-1 isoform has not yet been described for potato,
although the complex hybridization pattern of genomic DNA
blots suggests the presence of multiple PR-1 genes also in potato.
The accumulation of PR proteins is one of the most common
defence responses observed in the plant kingdom and, for the
majority of PR protein families, activities are known or can be
inferred (Kombrink and Somssich, 1997; van Loon and van Strien,
1999). Specific members of the tobacco and tomato PR-1 families
have been clearly demonstrated to be inhibitory to oomycete
pathogens (Alexander et al., 1993; Niderman et al., 1995), but
their mechanism of action remains completely unknown. Because
oomycetes contain little or no chitin in their cell walls, they
are not affected by chitinases. However, a search for proteins inter-
fering with their growth and development uncovered osmotin,
a salt stress-inducible protein of the PR-5 family from tobacco,
and related proteins from other plants as effective inhibitors
of Phytophthora infestans (Vigers et al., 1992; Woloshuk et al.,
1991). It was suggested that osmotin acts by permeabilizing
the membrane of pathogens. Both PR-1 and osmotin were
active not only in vitro, but also in vivo, when expressed at high
© 2002 BLACKWELL SCIENCE LTD MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345
Pathogenesis-related protein PR-1b from potato 339
levels in transgenic plants (Alexander et al., 1993; Velazhahan
et al., 1999).
Despite the proven potential of PR-1b to inhibit Phytophthora
infestans, all our data argue against a direct participation of
PR-1b in establishing race/cultivar-specific resistance in potato.
First, when incompatible and compatible interactions between
potato and Phytophthora infestans were compared, no significant
differences in the timing and magnitude of PR-1b induction
were observed at the early stages of infection (up to 3 days). This
holds true at the level of both protein accumulation and gene
activation. Furthermore, at late infection stages (4–5 days post-
inoculation), the protein amounts declined in the compatible
interaction, whereas they remained elevated in the incompatible
interaction. Second, when the temporal and spatial patterns of
PR-1b protein and mRNA accumulation within the infected tissue
were compared in compatible and incompatible interactions
by histological methods, no distinct differences could be observed
(see also below). Protein and mRNA distribution in the tissue
largely correlated with pathogen distribution. Third, we have
previously shown that race/cultivar specificity is apparently estab-
lished very early in the potato–Phytophthora infestans interaction
(Freytag et al., 1994; Schröder et al., 1992). The incompatible
interaction is characterized by the rapid appearance of fluoresc-
ing material, most probably wall-bound phenolics, in all plant
cells surrounding the invading pathogen. This autofluorescence
is the most rapid plant response to infection and is detectable
as early as 2 – 3 h post-inoculation. It occurs at drastically lower
frequency in the compatible interaction, indicating that the
pathogen escapes recognition or suppresses this initial defence
response. In any case, compared to this very rapid response,
changes in PR-1b expression were only detectable at relatively
late stages of the infection process.
Our results on PR-1b expression are in accordance with previ-
ously published data. van’t Klooster et al. (1999) recently cloned
a similar PR-1 isoform from potato and studied its expression in
response to infection by Phytophthora infestans. The accumula-
tion of PR-1 mRNA was similar in both the compatible and incom-
patible interactions up to 100 h post-inoculation, but thereafter
the mRNA amount declined in the compatible interaction, whereas
it remained elevated in the incompatible interaction. Again, a
decisive role of PR-1 in determining incompatibility cannot be
inferred from these results. For many other defence responses,
such as PAL, 4CL, 1,3-β-glucanase, chitinase, and GST1, we mostly
observed that the temporal and spatial expression patterns were
identical in compatible and incompatible interactions (Freytag
et al., 1994; Fritzemeier et al., 1987; Hahn and Strittmatter, 1994;
Kombrink et al., 1993; Schröder et al., 1992; Taylor et al., 1990).
Frequently, the induced mRNA and protein levels were higher
in the compatible interaction, presumably because of the larger
number of plant cells that respond to the ramifying virulent patho-
gen. All available results indicate that the difference between
MPP_126.fm Page 340 Wednesday, August 21, 2002 6:38 PM
340 E. HOEGEN et al.
resistance and susceptibility in the potato– Phytophthora infestans
interaction is of quantitative rather than qualitative nature despite
the presence of major resistance genes. It is the timing and
extent of hypersensitive response (HR) induction that seems to
play a decisive role (Freytag et al., 1994; Kombrink and Schmelzer,
2001; Vleeshouwers et al., 2000).
In contrast with the situation in potato, differential expression
of PR-1 homologues has been observed in other plant–pathogen
systems. In tomato, the accumulation of the PR-1 isoform, P14,
served as an early indicator of incompatibility on infection with
Cladosporium fulvum (syn. Fulvia fulvum), and a faster accumu-
lation occurred in the incompatible interaction (de Wit and van
der Meer, 1986; Joosten et al., 1990). Likewise, increased expres-
sion of PR-1 was also observed in resistant compared with near-
isogenic susceptible barley plants following inoculation with
Erysiphe graminis (syn. Blumeria graminis) (Muradov et al., 1993).
A differential response was observed in bell pepper ( Capsicum
annuum): faster and higher expression of PR-1, as well as gluca-
nase and chitinase, was observed in the incompatible interaction
with Xanthomonas campestris pv. vesicatoria, whereas with
Phytophthora capsici a strong PR-1 mRNA accumulation occurred
in both the compatible and incompatible interactions (Kim and
Hwang, 2000). Thus, the expression of PR-1 differs with the
experimental system and seems to depend on various factors,
including plant species, plant organ, and cell type, as well as the
mode of tissue colonization by the respective pathogens.
The analysis of the temporal and spatial patterns of PR-1b
expression by immunohistochemistry and in situ RNA hybridiza-
tion showed that the accumulation of PR-1b mRNA and protein
was clearly initiated at the infection site, but a delayed and
sustained synthesis was also observed in neighbouring, uninfected
potato leaves. Thus, the expression of PR-1b resembled that of
other PR proteins, such as 1,3-β-glucanases and chitinases (Ancillo
et al., 1999; Beerhues and Kombrink, 1994; Büchter et al., 1997;
Kombrink et al., 1993; Schröder et al., 1992). It is contrasted
by the rapid, localized and transient expression of other defence-
related genes, such as those encoding PAL, 4CL and GST1
(Freytag et al., 1994; Kombrink et al., 1993; Taylor et al., 1990).
PR-1b also displayed a strict development-, organ-, and cell
type-specific pattern of expression. The preferential accumulation
of mRNA and protein in the epidermis, vascular tissue, and guard
cells of the stomata is suggestive of a specific and important
function in these cells and in the plant’s defence programme
against pathogens. Stomata as natural openings in leaves are
the preferred, essential, or exclusive sites of entry into the leaf
for many pathogens, particularly downy mildews, rust fungi and
bacterial pathogens. Oomycetes, such as Phytophthora infestans,
enter the leaf mesophyll either via stomata or by penetrating
directly through the periclinal wall of an epidermal cell, and then
ramify mainly in the intercellular leaf space, sending haustoria
into mesophyll cells (Coffey and Wilson, 1983). The colocalization
MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345 © 2002 BLACKWELL SCIENCE LTD
of PR-1b and other defence proteins, such as chitinase (Büchter
et al., 1997), in guard cells may function as a preformed defence
barrier in this vulnerable position.
The induced accumulation of PR-1b in the epidermis further
underscores the importance of this outermost cell layer in the
defence programme. Again, PR-1b colocalizes in the potato leaf
epidermis with PAL and a specific, constitutively expressed,
chitinase isoform, as previously described (Ancillo et al., 1999;
Kombrink et al., 1993). Chitinases have also been reported to
preferentially accumulate in epidermal cells of tobacco and bean
leaves (Keefe et al., 1990; Mauch et al., 1992). Trichomes that
develop from epidermal cells differentially express cellular defence
responses. PR-1b can very clearly be localized in the cap structure
of glandular trichomes of potato leaves, together with PAL and
1,3-β-glucanase, as reported previously (Garcia-Garcia et al.,
1994). In contrast, chitinase is restricted to epidermal cells and
the basal cells of leaf hairs (Garcia-Garcia et al., 1994). In all
cases, a clear colocalization of mRNA and protein is observed.
Glandular trichomes are present on the foliage of many wild
and cultivated potato species, and the secreted material in their
glands contains different hydrophilic or lipophilic compounds,
such as sugars, polysaccharides, lipids, oils, resins, phenolic
compounds, and proteins, e.g. polyphenol oxidase and peroxidase
(Avé and Tingey, 1986; Ryan et al., 1982). Many of these have a
protective function against herbivores, pathogens, and environ-
mental stresses, such as extreme temperatures or extensive light
(Werker, 2000). The presence of PR-1b in the glandular trichomes
of potato adds to the defence potential against the pathogens of
these organs.
The accumulation of PR-1 and other PR proteins in vascular
bundles, especially the phloem, seems to be common for many
plants, including tobacco, tomato, and potato (Büchter et al.,
1997; Eyal et al., 1993; Tornero et al., 1997). It has been sug-
gested that the strong preference of PR-1 to accumulate in the
vascular tissue of pepper and tobacco stems may contribute to
the translocation of the protein throughout the plant (Eyal et al.,
1993; Lee et al., 2000). However, experimental evidence for this
proposal is lacking. The presence of PR-1b in the xylem and trac-
heids of potato is quite unexpected, because structural xylem
is a complex tissue consisting of different cell types, mostly non-
living cells, incapable of synthesizing new proteins (Esau, 1965).
The PR-1 proteins of tobacco are also localized in xylem elements
of infected leaves (Carr et al., 1987), and it has been suggested
that they enter the xylem via pits (pores) from the intercellular
fluid where they are quite abundant. However, experimental data
demonstrating that PR-1 proteins can be transported over con-
siderable distances through the xylem or phloem, for example to
other leaves, have not been presented.
From our histochemical analyses, we obtained unequivocal
evidence for PR-1b accumulation in specialized cells known as
crystal idioblasts. Interestingly, PR-1b protein was only detectable
MPP_126.fm Page 341 Wednesday, August 21, 2002 6:38 PM
in crystal idioblasts of infected potato leaves, whereas in unchal-
lenged leaves no staining of these cell types was observed. Other
PR proteins for which specific antisera were available, such as
1,3-β-glucanases (PR-2) and chitinases (PR-3), could not be
localized in crystal idioblasts, clearly demonstrating that crystal
idioblasts apparently selectively express and retain PR-1b.
There has only been one previous report demonstrating that PR-1
proteins are localized in crystal idioblasts; the protein was found
to accumulate in the vacuoles of these cells in tobacco leaves
(Dixon et al., 1991). Because the acidic PR-1 isoforms of tobacco
are normally secreted into the extracellular leaf space and not
retained in the vacuole, it has been proposed that crystal idio-
blasts utilize a unique and alternative cellular sorting mechanism
(Dixon et al., 1991). Whether potato crystal idioblasts also have
the capacity for de novo synthesis of PR-1b proteins is at present
unknown. In contrast with the results obtained with tobacco,
we have not been able to unequivocally localize PR-1b mRNA in
crystal idioblasts by in situ RNA hybridization. We attribute this
mainly to the fragile nature of these cells, which may have lost
their cellular content during the extensive tissue manipulation.
Thus, the function of PR-1b in crystal idioblasts and its precise
subcellular localization remain unknown. Crystal idioblasts are
most commonly associated with their ability to sequester Ca 2+ in
the form of calcium oxalate, and it has been proposed that they
may provide the plant with a mechanism for the regulation of
free Ca2+ levels. The accumulation of PR-1 proteins within these
cells during the infection process, and also during the subsequent
systemic acquired resistance response, might indicate a novel and
unique function of these cells in plant defence against pathogen
infection.
The three-dimensional structure of the tomato protein P14a
(PR-1b1), which is closely related to potato PR-1b, was recently
resolved by nuclear magnetic resonance spectroscopy (Fernández
et al., 1997). The protein has a unique molecular architecture,
which represents a rigid scaffold of tightly packed α-helices and
β-strands. This compact structure is further stabilized by the forma-
tion of disulphide bridges between the six conserved cysteine
residues, and it reflects the high stability of PR-1 proteins and
their insensitivity to several proteases. On the basis of the high
degree of sequence similarity, the three-dimensional fold of
tomato P14a can be expected to be common to all plant PR-1
proteins and similar proteins that have been found in fungi,
yeasts, insects, and vertebrates, including humans (van Loon
and van Strien, 1999). The human glioma pathogenesis-related
protein (GliPR), for example, which is highly expressed in the
brain tumour glioblastoma multiforme, exhibits 35% amino acid
sequence identity with tomato P14a (PR-1b1) and adopts the
same fold (Szyperski et al., 1998). Because of the strict conserva-
tion of the three-dimensional structure, as well as the geometry
of putative active site amino acid residues, it has been suggested
that human GliPR and plant PR-1 proteins may operate according
© 2002 BLACKWELL SCIENCE LTD MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345
Pathogenesis-related protein PR-1b from potato 341
to the same molecular mechanism. This could establish a possible
functional link between the human immune system and the plant
defence system (Szyperski et al., 1998). However, the nature of
the biochemical function of PR-1 and related proteins remains to
be uncovered.
E X P E R I M E N T A L P RO C E D U RE S
Plant material and protein extraction
Potato plants (Solanum tuberosum L. cvs. Datura and Désirée)
were grown from tubers in the greenhouse under controlled con-
ditions for 6 – 8 weeks as previously described (Kombrink et al.,
1988). The first to sixth fully expanded leaves (8–15 cm in length)
were used for the experiments. All stress treatments, including
inoculation with Phytophthora infestans, wounding, treatment
with fungal elicitor, ethylene, salicylic acid, or jasmonic acid,
were performed as previously described with detached leaves,
which were placed with their petioles into vials containing water
(Beerhues and Kombrink, 1994; Schröder et al., 1992).
The IWF and crude extracts of potato leaves were isolated as
previously described (Kombrink et al., 1988). Protein was deter-
mined according to Bradford (1976) using the Bio-Rad (München,
Germany) dye reagent and bovine serum albumin as standard.
Purification of potato PR-1 protein
All operations were carried out at 0 – 4 °C. Purification of the pre-
dominant extracellular PR-1 protein started from the IWF isolated
from 50 g of elicitor-treated potato leaves by infiltration with
40 mL H2O, yielding 40 mg of protein. After adjusting with 25 mM
sodium acetate (pH 5.0), the extract was passed through a CM-
cellulose column (2.5 × 30 mL CM 52; Whatman, Maidenhead,
UK; equilibrated with 25 mM sodium acetate, pH 5.0, 1 mM ethyl-
enediaminetetraacetic acid (EDTA), 0.5 mM dithiothreitol). After
washing with the same buffer, bound proteins were eluted with
a linear NaCl gradient (0 – 800 mM, 200 mL). Fractions containing
PR-1 were identified by SDS-PAGE, pooled, dialysed against
50 mM sodium formate (pH 4.5), and subjected to cation exchange
FPLC using a Mono S HR 5/ 5 column (Pharmacia, Uppsala,
Sweden) equilibrated with the same buffer. Bound proteins were
eluted with a linear NaCl gradient (0 – 400 mM, 20 mL) and
yielded two separate fractions containing PR-1 protein of appar-
ent homogeneity.
Antiserum
Rabbit antisera were produced by standard procedures (Hurn and
Chandler, 1980) using 100 µg of protein for the first injection and
50 µg of protein each for the subsequent three booster injections.
Two weeks after the last booster immunization, the serum was
MPP_126.fm Page 342 Wednesday, August 21, 2002 6:38 PM
342 E. HOEGEN et al.
collected. After clot removal, serum was clarified by centrifuga-
tion and stored in small aliquots at −80 °C.
Isolation and sequence analysis of PR-1b cDNA
Differential screening for pathogen-induced mRNAs resulted in
the isolation of several clones, including a small cDNA fragment
with high sequence similarity to proteins of the P14 family of
tomato (Tornero et al., 1993; van Kan et al., 1992). This cDNA
fragment was used to screen a cDNA library (lambda ZAP-Express,
Stratagene, La Jolla, USA) complementary to mRNA isolated
from systemically activated leaves of potato (cv. Désirée) following
local inoculation with Pseudomonas syringae pv. maculicola
(Hoegen, 1998). After three rounds of plaque purification, the
pBK-CMV plasmids harbouring EcoRI/XhoI cDNA insertions were
excised by in vivo zapping and used for sequence analysis and
expression studies.
DNA sequences were determined by the ADIS service unit
(Max-Planck-Institut für Züchtungsforschung, Köln, Germany) on
ABI PRISM 377 DNA Sequencers (PE Applied Biosystems, Foster
City, CA, USA) using BigDye Terminator chemistry. Sequences
were analysed on a UNIX computer network using the GCG (Genetics
Computer Group, Madison, USA) software package, version 10.0
(Devereux et al., 1984).
Heterologous expression of PR-1b in E. coli
For heterologous expression, the PR-1b coding sequence was
excised as a BamHI/SmaI fragment from the pBK-CMV cloning
vector and inserted into the E. coli expression vector pQE51
(Qiagen, Hilden, Germany), and the correct frame was confirmed
by sequence analysis. Proteins were expressed using E. coli strain
M15[pREP4] as recommended by the supplier (Qiagen, Hilden,
Germany). Cells were grown in 50 mL L-medium containing
100 µg/mL ampicillin and 25 µg/mL kanamycin at 37 °C (OD600 =
0.7 – 0.9). The addition of 2 mM IPTG induced the expression of
the protein and, after further incubation for 5 h at 37 °C, cells
were harvested by centrifugation. The pellet was resuspended in
3 mL buffer (50 mM sodium phosphate, pH 7.8, 0.3 M NaCl, 1 mg/
mL lysozyme) and, after 30 min incubation on ice, bacteria were
sonicated (8 × 15 s at 200–300 W with interruptions of 30 s).
Soluble and insoluble proteins were separated by centrifugation.
The pellet was resuspended in 0.4 mL SDS-PAGE sample buffer
containing 10% SDS and 5% 2-mercaptoethanol, and incubated
at 100 °C for 5 min. For SDS-PAGE analysis, this sample was 10-
fold diluted with sample buffer.
SDS-PAGE and Western blotting
Discontinuous SDS-PAGE and immunoblotting were carried out
as described previously (Kombrink et al., 1988). Proteins were
MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345 © 2002 BLACKWELL SCIENCE LTD
transferred electrophoretically at 30 V (=130 mA) overnight to
nitrocellulose sheets (BA 85; Schleicher & Schüll, Dassel, Germany)
in 25 mM tris glycine (pH 8.3) or 25 mM ethanolamine glycine
(pH 9.5), each containing 20% methanol. Peroxidase-conjugated
swine antibody to rabbit IgG (Dakopatts, Hamburg, Germany)
was used as a second antibody. Blots were developed in a mixture
containing 0.018% 4-chloro-1-naphthol, 6% methanol, and
0.012% H2O2 in PBS (137 mM NaCl, 3 mM KCl, 9 mM sodium
phosphate, pH 7.4). The peroxidase reaction was terminated
after 5– 10 min by several washes with water.
RNA and genomic DNA isolation and blot hybridization
Total RNA and genomic DNA from plants were isolated, denatured,
separated on agarose gels, and transferred to nylon membranes
(Hybond-N, Amersham Pharmacia Biotech, Freiburg, Germany),
as previously described (Ancillo et al., 1999). All DNA probes
were radiolabelled with [α-32P]dCTP (3000 Ci / mmol; Amersham
Pharmacia Biotech, Freiburg, Germany) using a random-primed
DNA labelling kit (Roche Diagnostics, Mannheim, Germany);
blots were hybridized at 60 °C and washed three times in
0.2 × SSC, 0.5% SDS at 60 °C.
Immunohistochemistry
For immunohistochemical analyses, the first fully expanded
leaves from the third internode were used. At various times after
inoculation, leaves were fixed by vacuum infiltration of a solution
containing 2% formaldehyde, 0.4% glutaraldehyde, and 5%
dimethyl sulphoxide in 0.1 M sodium phosphate (pH 7.0), and
the samples were left for 4 h on ice. The fixed tissue was washed
in 0.1 M sodium phosphate (pH 7.0), dehydrated in a series of
ethanol solutions in the same buffer (5 min each in 30, 50, 70, 85,
95 and 100% ethanol at 0 °C), and embedded in epoxy resin
(Kulzer and Co, Wehrheim, Germany), according to the manu-
facturer’s protocol. Embedded tissue was sectioned at 2 µm thick-
ness with a manual microtome (Leitz 1512, Wetzlar, Germany).
Sections were transferred to water droplets on microscope slides
for relaxation. After careful water removal, the slides were
exposed for 30 min to 60 °C on a heating plate. Immunohisto-
chemical treatments were carried out as described previously
(Schröder et al., 1992) with minor modifications. Prior to immune-
peroxidase staining, sections were incubated for 20 min with
80% methanol containing 3% H 2O2 to inactivate plant endo-
genous peroxidases. Subsequently, sections were incubated with
polyclonal antiserum raised against purified potato PR-1 protein
(preimmune serum served as control) or cell walls of Phyto-
phthora sp. (Jahnen and Hahlbrock, 1988), both diluted 1 : 200 in
PBS (150 mM NaCl, 25 mM sodium phosphate, pH 7.4) containing
1% bovine serum albumin. After three washes in PBS supple-
mented with 0.35 M NaCl and 0.1% Tween 20 (Serva, Heidelberg,
MPP_126.fm Page 343 Wednesday, August 21, 2002 6:38 PM
Germany), sections were incubated for 1 h at 37 °C with peroxidase-
conjugated goat immunoglobulins to rabbit immunoglobulins
(Dakopatts, Hamburg, Germany), diluted 1 : 100 in PBS contain-
ing 1% bovine serum albumin and 0.1% Tween 20. Follow-
ing three additional washes as above (supplemented PBS),
peroxidase activity was visualized by incubating tissue sections
with 1 mg/mL 3,3′-diaminobenzidine, 0.06% H2O2, 50 mM tris-
HCl (pH 7.6) at room temperature. Extensive washing with water
terminated the staining reaction. Tissue sections were mounted
in 10% Moviol 4-88 (Hoechst, Frankfurt, Germany), according to
the manufacturer’s conditions, and viewed with a Zeiss IM 35
microscope or Zeiss Axiophot microscope.
In situ RNA hybridization
For in situ mRNA localization, the first and second fully developed
leaves were used. At various time points after inoculation with
Phytophthora infestans, the leaf tissue was fixed in a solution
containing 4% formaldehyde, 0.25% glutaraldehyde, and 100 mM
sodium phosphate (pH 7.0) for 16 h at room temperature. After
dehydration in a series of ethanol solutions (see above), followed
by incubation in xylene, the tissue was infiltrated and embedded
in Histowax (Histolab, Göteborg, Sweden). Sections of 5 – 6 µm
thickness were prepared, put on aminopropyltriethoxy silane-
treated slides, and handled as previously described (Cox and
Goldberg, 1988), with the exception that the bovine serum
albumin treatment was omitted. After incubation with pro-
teinase K (2 mg/mL, 30 min at 37 °C) and glycine (2 mg/ mL in
200 mM sodium phosphate, pH 7.0), sections were hybridized
with sense (control) or antisense RNA probes, which were gener-
ated from linearized plasmid with T3 or T7 polymerase and the
DIG RNA labelling Kit, SP6 / T7 (Roche Diagnostics, Mannheim,
Germany), according to the manufacturer’s protocol. Hybridization
and washing conditions were as previously described (Cox
and Goldberg, 1988), with a final washing step in 0.1 × SSC.
After hybridization, the samples were treated with RNase A
(100 µg/mL, 30 min at 37 °C), followed by 20% normal sheep
serum (40 min), and incubated with the antidigoxigenin alkaline
phosphatase conjugate for 24 h at room temperature. The sub-
sequent enzyme-catalysed staining reaction was performed with
5-bromo-4-chloro-3-indolyl phosphate (X-phosphate) and freshly
prepared nitroblue tetrazolium (NBT) solution (75 mg/mL in 70%
dimethylformamide), according to the manufacturer’s protocol.
Sections were viewed with a Zeiss IM 35 microscope or Zeiss
Axiophot microscope.
ACKNOWLEDGEMENTS
We thank Drs Imre E. Somssich and Elmon Schmelzer for valuable
discussions and critical reading of the manuscript, and Brigitte
Pickel and Roswitha Lentz for their skilful technical assistance.
© 2002 BLACKWELL SCIENCE LTD MOLECULAR PLANT PATHOLOGY (2002) 3(5), 329 –345
Pathogenesis-related protein PR-1b from potato 343
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