Physiological and Molecular Plant Pathology (2002) 61, 227±236

doi:10.1006/pmpp.2002.0435, available online at http://www.idealibrary.com on

Hydrogen peroxide plays a critical role in the defence response of tomato

to Cladosporium fulvum

S T E P H A N IE B O R D E N * and V E R N A J . HI G G I N S {
Department of Botany, University of Toronto, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada

(Accepted for publication 12 August 2002)

The presence and localization of hydrogen peroxide (H2O2) in compatible and incompatible interactions
of Cladosporium fulvum and tomato were studied by light microscopy using the histochemical stain 3, 30 -
diaminobenzidine (DAB). To obtain synchronous development of the fungus, inoculation of tomato was
by injection of conidia into the intercellular area of the spongy mesophyll. Conidia were suspended in
scavengers of reactive oxygen species (ROS), i.e., catalase (CAT), superoxide dismutase (SOD), and
mannitol, or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, diphenylene
iodonium (DPI), at the time of inoculation. Conidia and initial germ tubes in compatible and
incompatible interactions caused a localized host reaction characterized by H2O2 production and a
variety of plant responses which included protein cross-linking, callose deposition, and accumulation of
phenolic compounds. Further growth of an avirulent race was restricted by similar host responses whereas,
hyphae of a virulent race grew as in a normal compatible interaction. In the incompatible interaction,
treatment with CAT, SOD, or DPI generally resulted in increased fungal length and a decrease in the
percentage of sites with host responses. These results support previous suggestions for this host±pathogen
system of a critical role for ROS in limiting colonization, and this appears to be partly as a signal molecule
for plant defence responses. *c 2002 Elsevier Science Ltd. All rights reserved.

Keywords: reactive oxygen species; catalase; superoxide dismutase; callose; cross-linked proteins;
phenolics; Lycopersicon esculentum; disease resistance.

INTRODUCTION occurring following perception of an avirulent pathogen

The Cladosporium fulvum±tomato interaction is a well-
described model system to study plant±pathogen inter-
actions [20]. This interaction is based on a typical
gene-for-gene relationship, i.e. for each dominant resis-
tance (R) gene present in the plant, there is a matching
dominant avirulence (Avr) gene present in the fungus [12].
Resistance in a gene-for-gene interaction occurs when an
elicitor, the product of an Avr gene in the pathogen, and
the product of the corresponding resistance gene in the
plant, interact directly or indirectly and trigger the
activation of an array of plant defence responses which
result in an incompatible interaction [22]. Plant responses
* E-mail: stephborden@hotmail.com
{ To whom all correspondence should be addressed. E-mail:
higgins@botany.utoronto.ca
Abbreviations used in the text: CAT, catalase; DAB, 3, 30 -
diaminobenzidine; DIC, di€erential interference contrast;
DMSO, dimethyl sulfoxide; DPI, diphenylene iodonium;
MES, 2-[N-morpholino] ethanesulfonic acid; NADPH, nicoti-
namide adenine dinucleotide phosphate; NBT, nitroblue tetra-
zolium; p.i., post inoculation; ROS, reactive oxygen species;
SDS, sodium dodecylsulphate; SOD, superoxide dismutase.
0885-5765/02/$ - see front matter
by host cells include reinforcement of the cell wall,
production of potentially antimicrobial compounds such
as phytoalexins and pathogenesis-related (PR) proteins,
cell death, and induction of the rapid generation of
reactive oxygen species (ROS). The C. fulvum±tomato
interaction is typical of this model and all of the typical
defence responses have been characterized, although not
all incompatible interactions result in cell death [14].
Although, binding of elicitor by the R gene product is
suggested, there is no evidence for speci®c binding
between Avr and resistance gene products in the
C. fulvum±tomato interaction [28].
One of the earliest plant responses to pathogens is the
rapid generation of ROS, such as O2 , H2O2, and HO.
[3, 39]. Various enzyme systems have been proposed as
the source for ROS in plants including a pH-dependent
cell wall peroxidase and/or a nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase system similar
to the system found in mammalian neutrophils [4, 26,
44]. In the C. fulvum±tomato interaction, there is evidence
that suggests a NADPH oxidase system is responsible for
production of ROS [36, 46].
*
c 2002 Elsevier Science Ltd. All rights reserved.
228 S. Borden and V. J. Higgins
Accumulation of ROS after pathogen attack is proposed
to contribute to a variety of types of defence, including
functioning as an antimicrobial compound [27, 35, 45], as
a facilitator of the formation of cell wall barriers through
oxidative cross-linking of cell wall structural proteins
[5, 6], as an inducer of host cell death [25], and as a
secondary signal molecule to activate further defence
responses [23, 25].
Most studies investigating the production of ROS in the
C. fulvum±tomato interaction have used race-speci®c
elicitors to treat suspension cultured tomato cells [42],
cotyledons [29], or whole leaves [27]. In such studies, host
cells are instantly and simultaneously ``assaulted'' by a
relatively high concentration of elicitor. These studies do
not adequately re¯ect the possible role of ROS in the actual
plant±pathogen interaction where elicitor concentrations
presumably build up gradually and are very localized.
The asynchronous nature of stomatal penetration by
C. fulvum [24] has made it dicult to quantitate the
frequency of ROS accumulation and other plant
responses that occur. Although De Wit [9] suggested
that guard cells were necessary for initiating the resistant
response in the C. fulvum±tomato interaction, Higgins
[16] showed that the expected disease reactions occurred
when tomato lea¯ets were inoculated by injection of
conidia to bypass stomatal penetration. Intercellular
injection of conidia of both virulent and avirulent races
of C. fulvum into leaf mesophyll induces localized
responses such as cell wall appositions and cell browning
[16]. Germ tubes of virulent races, however, soon grow
without causing a visible response in mesophyll cells, and
these hyphae continue to colonize host tissue without
further induction of defence responses. In contrast, the
growth of avirulent races is quickly arrested [16].
In the present study, the production of ROS was
examined in planta in tomato plants injection-inoculated
with conidia of C. fulvum with a variety of pharmacologi-
cal treatments designed to reduce the amount of O2 or
H2O2. The e€ect of such treatments on the extent and
localization of H2O2, on fungal growth, and on host cell
wall responses was determined.
MATERIALS AND METHODS
Plant and fungal material
Seeds of tomato cv. Moneymaker (no known genes for
resistance to C. fulvum and commonly referred to as Cf-0)
and near-isogenic line Cf-9, produced by multiple sel®ng
of a line established via ®ve backcrosses, were originally
obtained from I. Boukema, Institute for Horticulture
Plant Breeding, Wageningen, The Netherlands. Seeds
were germinated in 9-cell plastic packs containing
sterilized soil (Promix, general purpose growing medium,
Premier Horticulture Inc., PA, U.S.A.). Plants were
grown in a growth room with a 16 h photoperiod
(200 mE m 2 s 1) and a temperature of 228C.
Race 2.3 of C. fulvum (isolate # 54UT) used in this study
was originally isolated from cv. Vendor in a southern
Ontario greenhouse in 1980, and maintained at 808C in
50 % glycerol. Fungal cultures were subcultured on V-8
juice agar [32] and maintained at 228C with a 16 h
photoperiod (200 mE m 2 s 1).
Plant inoculation
For cotyledon inoculation, conidia from 14 day old
C. fulvum cultures were prepared in sterile 0.05 %
Tween-20 in water and washed by centrifugation three
times with the ®nal suspension (105 conidia ml 1) in
appropriate bu€er with or without pharmacological
treatments to reduce ROS. Conidial suspensions were
injected into the abaxial surface of cotyledons of 14 day
old tomato plants using a sterile 30 G1/2 needle on a 1 cc
syringe. Alternatively, cotyledons were spray-inoculated
with an atomizer to deliver conidia onto the cotyledon
surface. Inoculated plants were maintained in a high
humidity environment at 228C for the duration of the
experiment, generally 24 or 48 h p.i.
Pharmacological treatments
All solutions for pharmacological treatments were freshly
prepared before each experiment, and all were from Sigma
Co. (St. Louis, MO, U.S.A.) unless stated otherwise.
Conidia for injection were suspended in: 1100 U ml 1
CAT (CAT, from Aspergillis niger, EC 1.11.1.6) in 10 mM
2-[N-morpholino] ethanesulfonic acid (MES) bu€er, pH
6.5; 900 U ml 1 superoxide dismutase (SOD, from bovine
erythrocytes; ICN, EC 1.15.1.1) in MES bu€er;
1100 U ml 1 CAT ‡ 900 U ml 1 SOD in MES bu€er;
5 mM mannitol in MES bu€er; 5 mM DPI in 0.05 %
dimethyl sulfoxide (DMSO; Sigma). The control for all
experiments was 10 mM MES bu€er except for experi-
ments with DPI where 0.05 % DMSO was used.
Histological examination
Cotyledons were removed at the petiole at 12, 24, or 48 h
p.i. and were ®xed and decolourized in boiling 95 % (v/v)
ethanol, cleared in chloral hydrate (2.5 g ml 1), mounted
on glass slides in modi®ed Hoyer's medium [38], and
examined with an Olympus Provis AX70 microscope
(Olympus America, Inc., Melville, New York, U.S.A.)
equipped with di€erential interference contrast (DIC)
optics. Digital micrographs were captured with a Spot
digital camera (Diagnostic Instruments Inc., Stearling
Heights, MI, U.S.A.) and processed with Image Pro Plus
software (Media Cybergenetics, Silver Spring, MD,
 Hydrogen peroxide role in response to Cladosporium fulvum
U.S.A.). For each experiment, 25 sites were examined
from four separate tomato cotyledons from four di€erent
plants (i.e. n ˆ 100). Only injected conidia that were
deposited beyond the initial wound site were included in
recorded observations. Hyphal length and the presence or
absence of visible plant responses associated with host cell
wall±hyphae contact sites were recorded for each site in
the treatments. Uninoculated controls were not necessary
because of the restriction of observations to wall±hyphae
contact sites, and as injected areas in which conidia were
absent rarely showed staining or other visible responses.
Detection of hydrogen peroxide and superoxide
To localize H2O2 and O2 in tomato cotyledons in
response to inoculation, an aqueous solution (2 mg ml 1)
of 3,30 -diaminobenzidine (DAB) in MES bu€er or 0.5 %
nitroblue tetrazolium (NBT) in MES bu€er was injected
into the abaxial surface of tomato cotyledons 2 h or
30 min before sampling, respectively [15]. Hence, the
stains detected the H2O2 and O2 present when the stains
were injected and that accumulating over the next
30 min …O2 † or 2 h (H2O2). Tissue was cleared and
processed as above for histological examination.
Detection of cross-linked cell wall proteins, callose,
and phenolics
At 12, 24, or 48 h p.i., inoculated cotyledons were pre-
pared for detection of protein cross-linking, callose
deposition, or phenolic accumulation. To visualize infec-
tion sites with H2O2-induced cross-linked proteins, a
modi®ed version [31] of the procedure described by
Thordal-Christensen et al. [41] was used. At various times
p.i., injected cotyledons were ®xed and decolourized in
boiling 95 % (v/v) ethanol and then, to remove soluble
proteins, were submerged in 1 % SDS for 3 days at 808C.
The SDS-insoluble proteins were subsequently stained
with 0.1 % (w/v) Coomassie blue (Consolidated Labora-
tories, Inc., Weston, ON, Canada) in 40 % (v/v) ethanol
and 10 % (v/v) acetic acid for 20 min, followed by
destaining in 40 % (v/v) ethanol and 10 % (v/v) acetic
acid. Tissue was mounted in the destaining solution for
light microscopy. The number of cell wall±hyphae
contact sites with dark blue colour, indicating cross-linked
proteins, was recorded.
To detect callose at infection sites, inoculated cotyle-
dons were ®xed and decolourized in boiling 95 % (v/v)
ethanol, then the tissue was submerged in 0.1 M KPO4
bu€er, pH 9.0, for 24 h followed by staining in 0.01 %
aniline blue (Fisher Scienti®c Co., Fair Lawn, NJ,
U.S.A.) in KPO4 bu€er for 2 h. Tissue was mounted in
the KPO4 bu€er and examined under blue light
epi¯uoresence. Areas of callose deposition ¯uoresce bright
yellow. For some experiments, the presence of callose and
229
cross-linked proteins was detected simultaneously in
inoculated tissue by staining the SDS treated Coomassie
blue stained cotyledons as above with aniline blue. The
number of cell wall±hyphae contact sites with callose,
and with both callose and cross-linking was recorded.
Phenolic compounds were detected by toluidine blue
using a protocol by O'Brien et al. [34]. Inoculated
cotyledons were ®xed and decolourized in boiling 95 %
(v/v) ethanol then submerged in 50 mM citrate bu€er pH
3.5, for 1 h, followed by staining with 0.05 % toluidine
blue (Matheson Coleman and Bell, Norwood, OH,
U.S.A.) in citrate bu€er for 20 min. Phenolic compounds
were visualized as blue/turquoise staining. The number of
cell wall±hyphae contact sites with phenolic compounds
was recorded.
Statistical analysis
The data presented represent means of hyphal lengths or
percentages of cell wall±hyphae contact sites calculated
from 25 reaction sites on each of four cotyledons per
treatment. Where necessary, the arcsine transformation
was used to normalize data. Comparisons of di€erences
observed in all experiments were made using the Student's
t-test where P 4 0.05 was considered signi®cant. All
experiments were performed twice with similar results;
typical data from one experiment are shown in each ®gure.
RESULTS
Con®rmation of compatible and incompatible interactions on
injection inoculation
Inoculation by means of injection of conidia of C. fulvum
was a useful technique to ensure synchronous conidial
germination and fungal growth in tomato cotyledons.
After inoculation of Cf-0 and Cf-9 near-isogenic lines of
tomato cv. Moneymaker with conidia of C. fulvum race 2.3,
the compatible and incompatible interactions were
observed, respectively. Macroscopic signs of disease in
the compatible interaction were evident as early as 10 days
after inoculation and included areas of densely sporulating
mycelium on the abaxial surface of tomato cotyledons.
Sporulation, or any other macroscopic evidence of
infection, was absent on cotyledons in the incompatible
interaction (results not shown). Microscopic examination
of individual conidia at 24 h p.i. was used routinely as
most conidia had germinated but hyphal growth was still
limited enough to measure. Sampling at 12 h p.i. was also
used in initial experiments to detect early production of
H 2O 2 .
230 S. Borden and V. J. Higgins
Localization of reactive oxygen species
No localized NBT staining, indicative of the accumulation
of O2 , was observed in cotyledons at the time points
(12 and 24 h p.i.) examined in either the compatible or
incompatible interaction (results not shown). In tissue
with DAB staining to detect H2O2, a reddish-brown to
yellow precipitate was present at plant cell wall apposi-
tions that had formed near conidia or germ tubes
[Fig. 1(a) and (b)]. This staining was con®rmed to be
speci®c for H2O2 by co-injection of DAB and CAT into
inoculated tomato cotyledons 2 h before sampling. This
co-injection signi®cantly decreased the percentage of wall
appositions with DAB staining in both the compatible and
incompatible interactions (results not shown). A test for
the presence of adequate peroxidase activity, to facilitate
the DAB reaction in the cotyledons, involved co-injection
of uninoculated tissue with DAB and 0.1 M H2O2. In such
cotyledons, DAB staining was seen along the epidermal
cell wall. In a water control, i.e. DAB-minus inoculated
cotyledons, no colouration comparable to that seen with
DAB was observed at wall appositions near hyphae.
As early as 12 h p.i., wall appositions with DAB
staining were present where the initial germ tubes
contacted the epidermal or mesophyll plant cell wall.
These DAB-stained wall appositions were seen as
anticlinal wall thickenings, or as halos if seen on the
periclinal walls [Fig. 1(a)]. In both interactions most of
the initial germ tubes appeared to stop growing, but
additional germ tubes developed in the compatible
interaction, and these generally grew without causing
wall appositions or DAB staining and generally appeared
robust [Fig. 1(a)]. In the incompatible interaction,
additional germ tubes generally caused host reactions
with DAB staining similar to that seen with initial germ
tubes [Fig. 1(b)]. For clarity, these additional germ tubes
will be termed the secondary germ tubes to distinguish
them from the initial ( primary) germ tubes. The data,

F I G . 1. Light micrographs (DIC optics) of tomato cotyledons injection-inoculated with race 2.3 of C. fulvum illustrating various
plant responses at cell wall±hyphae contact sites in compatible (Cf-0) and incompatible (Cf-9) lines. (Bars, 20 mm). DAB staining,
indicative of H2O2 (small arrowheads), is present at some cell wall±hyphae contact sites in both compatible (a) and incompatible
(b) interactions but was not present around a robust hypha (large arrowhead) in the compatible interaction. Accumulation of
phenolics (c) at a contact site as detected by toluidine blue staining (arrowhead) in an incompatible interaction. Protein cross-
linking (d) as detected by Coomassie blue (arrowheads) is present in the incompatible interaction at contact sites. The presence of
callose (e) in the same tissue as in (d) was indicated by the ¯uorescence of aniline blue under blue light.
 Hydrogen peroxide role in response to Cladosporium fulvum 231
however, for wall±hyphae contact sites include both 80
(a) Cf–0 12 hpi (b) Cf–9 12 hpi
primary and secondary hyphae.

Wall-hyphae contact sites with

60

40
* * *
E€ect of ROS scavengers and an NADPH oxidase inhibitor

DAB stain (%)

20
on ROS accumulation and fungal growth
0
The importance of ROS accumulation in limiting the 80
(c) Cf–0 24 hpi (d) Cf–9 24 hpi
growth of C. fulvum in tomato cotyledons was examined 60
by co-injection of conidia with CAT to reduce H2O2, * * *
SOD to remove O2 , CAT and SOD combined, the HO. 40 * *
scavenger mannitol, and the NADPH oxidase inhibitor 20
DPI into Cf-0 and Cf-9 tomato cotyledons. 0
In the compatible interaction on Cf-0, no signi®cant MES CAT SOD CAT+ MES CAT SOD CAT+
SOD SOD
increases in fungal length, as compared to the MES
F I G . 3. The e€ect of the ROS scavengers CAT and SOD on the
control, were observed at 12 or 24 h p.i. in any treatments percentage of cell wall±hyphae contact sites with DAB staining in
[Fig. 2(a) and (c)]. The percentage of hyphae associated compatible (a) and (c) and incompatible (b) and (d) interactions
with DAB staining at cell wall±hyphae contact sites of C. fulvum and tomato at 12 and 24 h p.i. At inoculation, conidia
signi®cantly decreased in SOD and CAT ‡ SOD treat- were suspended in: 1100 U ml 1 CAT in 10 mM MES bu€er,
ments at 24 h p.i. [Fig. 3(c)] but no di€erences were seen 900 U ml 1 SOD, or 1100 ‡ 900 U ml 1 CAT ‡ SOD all in
at 12 h p.i. [Fig. 3(a)] when only a low percentage of MES bu€er. 2 h prior to harvest, DAB (2 mg ml 1) was injected
into cotyledons. Each bar is the mean + SD of four replicates,
sites were DAB-stained. The lag in the appearance of
with 25 sites each. An * indicates that values di€er signi®cantly
DAB staining at sites where hyphae contact the cells, as from the MES control at P 4 0.05.
compared to the incompatible interaction [Fig. 3(b) and
(d)], suggests slower accumulation of H2O2 at these sites
which predominantly involve primary hyphae. At 24 h
p.i., no signi®cant di€erences were seen in mean fungal the percentage of DAB-stained wall appositions near
length in the mannitol treatment, as compared to the hyphae [Fig. 4(c)] as compared to the DMSO control.
control (27.7 + 1.7 and 32.0 + 3.0 mm in control and In the incompatible interaction on Cf-9, no signi®cant
mannitol treatments, respectively). By 12 h p.i., di€erences in fungal length were observed at 12 h p.i., as
DPI treatment of the compatible interaction had no compared to the MES control, however, at 24 h p.i.,
signi®cant e€ect on hyphal length [Fig. 4(a)] or on signi®cant increases in fungal length were observed in all

60
Length of hyphae

(a) Cf–0 (b) Cf–9

*
100 (a) Cf–0 12 hpi (b) Cf–9 12 hpi
(µm)

40
80
Mean length of hyphae (µm)

60 20
∗
40
0
20
sites with DAB stain (%)
Wall-hyphae contact

0 (c) Cf–0 (d) Cf–9

30
100 (c) Cf–0 24 hpi (d) Cf–9 24 hpi
20
80 * * *
*
60 10
40
0
20 DMSO DPI DMSO DPI

0
MES CAT SOD CAT+
SOD
MES
F I G . 2. The e€ect of the ROS scavengers CAT and SOD on
length of hyphae in compatible (a) and (c) and incompatible (b)
and (d) interactions of C. fulvum and tomato at 12 and 24 h p.i. At
inoculation, conidia were suspended in: 1100 U ml 1 CAT in
10 mM MES bu€er, 900 U ml 1 SOD, or 1100 ‡ 900 U ml 1
CAT ‡ SOD all in MES bu€er. Each bar is the mean + SD of
four replicates, with 25 sites each. An * indicates that values di€er
signi®cantly from the MES control at P 4 0.05.
CAT SOD CAT+
SOD F I G . 4. The e€ect of the NADPH oxidase inhibitor DPI on
length of hyphae (a) and (b) and the percentage of cell wall±
hyphae contact sites with DAB staining (c) and (d) in
compatible (a) and (c) and incompatible (b) and (d)
interactions of C. fulvum and tomato at 12 h p.i. At inoculation,
conidia were suspended in: 0.05 % DMSO or 5 mM DPI in
DMSO. 2 h prior to harvest DAB (2 mg ml 1) was injected
into cotyledons. Each bar is the mean + SD of four replicates,
with 25 sites each. An * indicates that values di€er signi®cantly
from the DMSO control at P 4 0.05.
232
treatments [Fig. 2(b) and (d)]. At 12 h p.i. DAB staining
[Fig. 3(b)] was strikingly more conspicuous than in the
compatible interaction [Fig. 3(a)] and the percentage of
DAB-stained sites decreased signi®cantly at both 12 and
24 h p.i. in all treatments [Fig. 3(b) and (d)]. In the
mannitol treatment, at 24 h p.i. a small signi®cant
di€erence was observed in mean fungal length
(19.7 + 2.2 mm and 26.0 + 4.3 mm in control and
mannitol treatments, respectively). Hyphae in DPI
treatments grew signi®cantly more [Fig. 4(b)] and were
associated with signi®cantly fewer DAB-stained sites, as
compared to the DMSO control [Fig. 4(d)].
Cross-linking of plant cell wall proteins
Cross-linking of plant cell wall proteins to strengthen the
wall has been suggested to be a defence response that is
mediated by H2O2 [5, 6]. Protein cross-linking was
examined in CAT- or SOD-treated, and MES control
tissue. After treatment of inoculated tomato cotyledons
with SDS to remove soluble cell wall proteins, and
staining with Coomassie blue, the remaining insoluble
cross-linked proteins were seen microscopically as blue
stained areas at wall appositions [Fig. 1(d)]. In the
compatible interaction, CAT and SOD did not cause a
signi®cant decrease in the percentage of reaction sites
with callose (%) with cross-linking (%)
S. Borden and V. J. Higgins
with protein cross-linking at 24 h p.i., as compared to the
MES control [Fig. 5(a)]. In the incompatible interaction,
the percentage of reaction sites with protein cross-linking
was markedly higher than in the compatible and was
signi®cantly reduced in both CAT and SOD treatments
at 24 h p.i., as compared to the MES control [Fig. 5(b)].
Tomato cotyledons that were spray inoculated, i.e. not
inoculated by means of injection, showed protein cross-
linking reactions in compatible (8 % of stomata) and
incompatible (66 % of stomata) interactions after stoma-
tal penetration but, because the SDS treatment to remove
soluble proteins severely disrupts the integrity of the
cotyledon surface, only 50 penetration sites for each
interaction were observed.
Callose and cross-linked cell wall proteins at contact sites
Callose and cross-linked proteins were examined in the
same tissue at 24 h p.i. using aniline blue and Coomassie
blue, respectively [Figs 1(d), (e) and 5]. Wall appositions
¯uoresced bright yellow in aniline blue-stained tissue
indicating the presence of callose, but was absent in
comparable tissue treated with bu€er alone. Callose was
detected at wall appositions in both compatible and
incompatible interactions. In the compatible interaction,
the ROS scavengers CAT and SOD did not signi®cantly

(b) Cf–9
80 (a) Cf–0
60 * *
40
Cell wall-hyphae contact sites

20
0

(c) Cf–0 (d) Cf–9

80
60
40 * *
20
0
80
with cross-linking
and callose (%)

(e) Cf–0 (f) Cf–9

60
40

20 * *

0
MES CAT SOD MES CAT SOD

F I G . 5. The e€ect of the ROS scavengers CAT and SOD on protein cross-linking (a) and (b), callose (c) and (d), and cross-linking
and callose (e) and ( f) at cell wall±hyphae contact sites in compatible (a), (c), and (e) and incompatible (b), (d), and ( f)
interactions of C. fulvum and tomato at 24 h p.i. At inoculation, conidia were suspended in 1100 U ml 1 CAT in 10 mM MES
bu€er, or 900 U ml 1 SOD in MES bu€er. At sampling time, tissue was prepared for detection of the various responses. Each bar is
the mean + SD of four replicates, with 25 sites each. An * indicates that values di€er signi®cantly from the MES control at
P 4 0.05.
 Hydrogen peroxide role in response to Cladosporium fulvum
decrease the percentage of infection sites with callose
alone at 24 h p.i. [Fig. 5(c)]; however, there was a
signi®cant reduction in the percentage of sites with
callose at 48 h p.i. in the SOD treatment, as compared to
the MES control (data not shown). In the incompatible
interaction, CAT and SOD treatments both signi®cantly
decreased the percentage of sites with callose almost
equally at 24 [Fig. 5(d)] and 48 h p.i. (data not shown).
Data on the co-occurrence of callose and cross-linked
proteins [Fig. 5(e) and ( f)], compared with the fre-
quency of occurrence of each [Fig. 5(a)±(d)], indicate
that in the compatible interaction roughly 50 % of
control sites with callose also had cross-linked protein;
in contrast, in the incompatible interaction about 75 % of
control sites with callose also contained cross-linked
protein. In the compatible interaction, treatment with
CAT or SOD did not substantially change the percentage
of callose sites with cross-linked proteins but the
incompatible interaction had markedly lower percentages
(CAT: ca 30 %, SOD: ca 50 %).
Phenolic compounds at contact sites
Toluidine blue staining indicating phenolic compounds
was generally detected adjacent to conidia [Fig. 1(c)] and
at wall appositions near fungal hyphae in tomato
cotyledons. Phenolic compounds were detected at the
same location as callose and cross-linked proteins and
were much less frequent in the compatible interaction
[Fig. 6(a)] than in the incompatible interaction
[Fig. 6(b)]. After treatment with CAT, there was no
signi®cant reduction in the percentage of compatible sites
with phenolic compounds at 24 h p.i., as compared to the
MES control [Fig. 6(a)]. In the incompatible inter-
action, however, there was a signi®cant reduction in the
percentage of sites with phenolic compounds with CAT
treatment [Fig. 6(b)].
sites with phenolics (%)
80
Wall-hyphae contact
(a) Cf–0 (b) Cf–9
60
40
20
0 In many plant±pathogen interactions ROS are pro-
MES CAT MES
F I G . 6. The e€ect of the ROS scavenger CAT on the percentage
of cell wall±hyphae contact sites with phenolic compounds in
compatible (a) and incompatible (b) interactions of C. fulvum
and tomato at 24 h p.i. At inoculation, conidia were suspended
in 1100 U ml 1 CAT in 10 mM MES bu€er. At sampling time,
tissue was prepared for detection of phenolics using toluidine
blue. Each bar is the mean + SD of four replicates, with 25 sites
each. An * indicates that values di€er signi®cantly from the
MES control at P 4 0.05.
233
DISCUSSION
In the C. fulvum±tomato interaction, it is clear that
following the interaction between the fungal AVR
product and the host Cf gene product, and subsequent
signalling events, many putative defences are activated.
Phytoalexin production [10, 11], synthesis of chitinases
and glucanases [21], cell death [14, 24], and the release of
ROS [27, 29, 42] are among the best characterized of
these defences. Yet, the extent to which any one of these
changes is involved in limiting growth of the fungus is
unknown.
The goal of this study was to determine if ROS
produced in the C. fulvum±tomato interaction are involved
directly or indirectly in restricting fungal growth. Previous
data obtained from elicitor treatment of cell suspension
cultures [42], cotyledons [29], and whole leaves [27]
suggested that ROS are involved in plant defence of
tomato to C. fulvum. However, such studies in which
elicitors are added once at a relatively high concentration
may be unlike the slower, presumably gradual, release of
elicitor by a germ tube. Here the extent and localization of
H2O2 production was facilitated by an inoculation
method whereby conidia were injected into tomato
cotyledons to circumvent the asynchronous nature of
stomatal penetration. Initial experiments con®rmed the
results of Higgins [16] that showed that race speci®city
was retained following injection inoculation. We com-
pared those histologically detectable events in compatible
and incompatible interactions that occurred following the
initial non-race speci®c host cell reaction to the conidia
and initial germ tube. Gene-for-gene speci®c signals
appear to control these subsequent e€ects. In this report,
C. fulvum race 2.3 (which produces AVR9) was inoculated
into the susceptible cv. Moneymaker (Cf-0) and the
resistant near-isogenic Cf-9 line. Other experiments with
race 2.4.5.9 (which lacks Avr9) on both Cf-0 and Cf-9
plants produced results similar to those reported for race
2.3 on Cf-0 plants (Borden and Higgins, unpublished
work). The conidial injection technique combined with
DAB staining allowed a pharmacological approach in
which scavengers of H2O2 (CAT) or O2 (SOD) could be
added to the apoplast at the time of inoculation to test
* their e€ect on H2O2 accumulation and hyphal growth
simultaneously.
CAT duced but the role they play in restriction of fungal
growth is not fully elucidated. Some evidence is provided
by the occurrence of microbial CATs, which act as
virulence factors during pathogenesis [13, 47]. Mutant
strains of Erwinia chrysanthemi de®cient in SOD activity
cause less disease on African violets than wild-type
bacteria indicating that SOD may also be a virulence
factor [37]. In addition, oxalic acid appears to be
required for e€ective pathogenesis in the Sclerotinia
234 S. Borden and V. J. Higgins
sclerotiorum±tobacco interaction by interfering with the
oxidative burst [7]. Cross-linking of plant cell wall
proteins, a process mediated by H2O2, also appears to
be involved in restricting fungal penetration of epidermal
cells in the barley powdery mildew±barley interaction
[41]. Mellersh et al. [31] found that H2O2 production was
the only response in three plant±fungi combinations to
occur at the appropriate time and place to account for the
failure of the fungi to penetrate. In one of these three
examples, the quiescent infection of tomato cotyledons by
Colletotrichum coccodes, the failure of the fungus to penetrate
was almost totally eliminated by intercellular injection of
CAT at the time of inoculation.
In this study, histochemical staining with DAB proved
to be an e€ective probe for H2O2 accumulation. As early
as 12 h p.i., DAB staining, indicative of H2O2, was
present at plant cell wall appositions formed adjacent to
conidia and germ tubes in both the compatible and
incompatible interactions. Similar DAB staining was seen
at host reaction sites in barley after inoculation with the
barley powdery mildew fungus [41] and in the three
interactions studied by Mellersh et al. [31].
In analysing the results of this study, it is important to
brie¯y consider the possible sources of H2O2. A NADPH
oxidase system similar to that of mammalian neutrophils
may be one source of ROS in plants [4, 43, 46]. In
contrast, in some plant±pathogen interactions, the source
of ROS appears to involve extracellular peroxidases via a
bound O2 intermediate [4]. Although NADPH oxidase,
the proposed source of H2O2 in the C. fulvum±tomato
interaction [17, 46], requires O2 as the initial ROS, our
attempts to localize O2 using NBT in cotyledons failed.
Failure to detect O2 , when it is a suspected precursor to
H2O2, has been observed in other plant±pathogen
interactions [19, 25]. In these interactions, O2 may be
rapidly dismutated to H2O2, which thus becomes the
major ROS that accumulates. After treatment with race-
speci®c elicitors of C. fulvum, NBT staining was detected
in incompatible but not compatible interactions of
tomato cotyledons at 2 h [29] and was one of the earliest
defence responses occurring. In addition, O2 has also
previously been detected biochemically in the C. fulvum±
tomato interaction using elicited-tomato suspension cells
[42] and localized histologically in elicitor-treated tomato
leaves [27]. In this study, inoculation of tomato
cotyledons with conidia suspended in the NADPH
oxidase inhibitor DPI resulted in a decrease in the
percentage of hyphae associated with DAB staining in the
incompatible interaction supporting the suggested [36,
46] role for NADPH oxidase, and/or other DPI sensitive
sources of H2O2, in this defence response. The failure of
DPI to further reduce the low percentage of DAB staining
sites in the compatible interaction suggests that the H2O2
produced on the initial encounter [16] of primary hyphae
with host cells may originate by DPI insensitive processes
[3, 43], i.e. the source(s) of H2O2 may di€er between the
host speci®c reaction and the early non-speci®c reactions
seen in the compatible interaction [16]. The slower rate at
which DAB staining occurred at these sites in the
compatible as compared to the incompatible interaction
also suggests the possibility of di€erent sources of H2O2.
There are many proposed roles for H2O2 in plant
defence responses including functioning as an antimicro-
bial compound, a signaling molecule [43], a facilitator of
formation of plant cell wall barriers, and an inducer of
cell death [3, 8, 23, 30, 39, 40]. In this study, in the
incompatible but not the compatible interaction, fungal
growth generally increased with either CAT, SOD or
DPI treatments, providing evidence for an e€ect of H2O2
and/or O2 in restricting fungal growth. Generally, the
increase in hyphal growth coincided with reduced DAB
staining at 24 h p.i., but at 12 h p.i. DAB staining was
reduced but hyphal growth was not. This may suggest
that 12 h p.i. was about the point when hyphal growth
was arrested in the incompatible interaction.
It should be noted that appropriate ``protein'' controls
for the experiments involving injection of CAT or SOD
into cotyledons do not exist. Denatured enzymes are
aggregated and less soluble than native proteins and thus
inappropriate. Non-denatured, inert, proteins such as
bovine serum albumin di€er from CAT and SOD in
amino acid content and tertiary structure and thus are a
poor substitute. In a related study [31] with three other
host±pathogen interactions, the e€ectiveness of CAT and
ine€ectiveness of SOD in reducing DAB staining and
increasing penetration success reduced the need for such a
control. Hence, in this study while it is unlikely that a
non-speci®c protein e€ect, e.g. fungal nutrition or
scavenging of ROS, could account for the results, that
possibility cannot be eliminated.
The failure of the combined CAT‡SOD treatment to
increase fungal growth or the percentage of hyphae
associated with DAB staining, as compared to the CAT or
SOD alone [Fig. 2(d)] suggests both CAT and SOD
a€ect the same phenomenon. If an NADPH oxidase
system is the source of most ROS in the C. fulvum±tomato
interaction, the addition of SOD to cotyledons should
increase the production of H2O2. However, an increase in
the percentage of reaction sites with DAB staining, as
compared to the MES control, was not observed. The
e€ect of SOD on the amount of OH., the most
antimicrobial ROS, could be a factor in the role of
SOD in increasing hyphal growth. In the presence of
Fe3‡ , O2 and H2O2 interact to produce OH. [3, 39].
If SOD reduces the amount of O2 , and consequently
causes a reduction in OH., hyphal growth would be less
restricted. This possibility is supported by treatments
with the OH. scavenger (mannitol), in which mean
fungal length increased signi®cantly in incompatible but
not compatible interactions. Additionally, it is possible
 Hydrogen peroxide role in response to Cladosporium fulvum
that SOD changes the kinetics of H2O2 accumulation and
degradation. For example, it was recently demonstrated
that horseradish peroxidase (an extracellular plant
enzyme) can act as a CAT [18] and that scavengers of
O2 , such as tetranitromethane and SOD, enhanced this
CAT-like activity. In the C. fulvum±tomato experimental
system, existing peroxidases, or ones induced in the
incompatible interaction, may catalyze a similar reaction
on the addition of SOD.
ROS induced plant responses that may be involved in
containment of fungal hyphae include cross-linking of cell
wall proteins, callose depositions, and accumulation of
phenolic compounds. These three features of wall
``strengthening'' were localized with H2O2 around conidia
and initial germ tubes in both compatible and incompa-
tible interactions but less frequently around the secondary
germ tubes formed later in the compatible interaction.
Although cross-linking of wall proteins to form an
insoluble protein barrier is a well-documented direct
e€ect of H2O2 accumulation [5, 6], the relationship of
callose deposition and phenolic compounds to H2O2
production is less clear. Again, the ability of CAT and
SOD to decrease H2O2 accumulation was used to test the
role of H2O2 as a signal for the cell wall changes. In the
incompatible but not the compatible interaction, the
percentage of wall appositions near fungal hyphae with
these three responses generally decreased signi®cantly, as
compared to the MES control, suggesting that H2O2 may
be a signal for these responses.
H2O2, cross-linking, callose, and phenolics were
localized at wall appositions near fungal hyphae which
suggest that these responses are involved in a coordinated
plant defence response. The percentage of appositions
with both callose and cross-linking was highest in
incompatible interaction suggesting a qualitative di€er-
ence in the composition of wall appositions with walls in
the incompatible interaction better ``armored''. Callose
deposition is a common localized response of plants to
attempted penetration and is often impregnated with
phenolic compounds, which may be involved in plant
defense [1, 2, 33]. As C. fulvum hyphae grow only in the
apoplast, these wall-associated responses should not
physically restrict fungal growth but such structural
changes may restrict the outward ¯ow of nutrients from
host cells and perhaps con®ne host (and fungal) products
to the immediate site, perhaps to the detriment of the
fungus.
The high frequency in incompatible interactions of
wall appositions with H2O2, cross-linking, callose, and
phenolics, and the fact that CAT or SOD generally
decreased these responses, support a major role of H2O2
in restricting fungal growth. A direct antimicrobial e€ect
of H2O2 cannot be determined as all of the responses
studied were generally localized at the same location at
about the same time. Probably the more important role
235
of H2O2 is as a signal {[43] and references therein}. This
experimental system may facilitate further pharmacologi-
cal approaches to determine if other putative defences are
involved in restricting the growth of C. fulvum.
The authors thank A. Cheung for expert technical
assistance. This work was supported in part by funds
from the Natural Sciences and Engineering Research
Council of Canada and by a University of Toronto Open
Fellowship to S.B.
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