Download Role of an endogenous nitric oxide burst in the resistance of wheat

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2004? 2004
274473477
Original Article
NO in the wheat-stripe rust system
P. Guo
et al.
Plant, Cell and Environment (2004) 27, 473–477
Role of an endogenous nitric oxide burst in the resistance
of wheat to stripe rust
P. GUO1,2,*, Y. CAO1,*, Z. LI2 & B. ZHAO1
1
Institute of Biophysics, Academia Sinica, 15 Datun Road, Chaoyang District, Beijing 100101, China and 2College of Plant
Protection, North-west Science-Technology University of Agriculture and Forestry, Yangling, China 712100
ABSTRACT
INTRODUCTION
The stripe rust (or yellow rust) disease caused by Puccinia striiformis Westend is a serious disease of wheat in
many areas of the world. The role of NO, which is an
important redox-active signalling molecule in plants, was
investigated in the wheat-stripe rust system. The phenotypes from interactions of the same wheat variety,
Lovrin10, with two different clones of stripe rust strains
(P. striiformis Westend), namely China yellow rust
(CY)22-2 and CY29-1, which are immune and susceptible
reaction types, respectively. The time course of host
endogenous NO detected by electron spin resonance indicated that recognition of an avirulent strain was associated with two peaks of NO production. The first peak of
NO accumulated in the early infection stage whereas the
second peak accumulated in the latent period; however,
only a single peak of NO was observed in the latent
period for the virulent strain. Furthermore, the activity of
pathogen-related protein-phenylalanine ammonia-lyase
was higher in the resistant system than in the susceptible
system, which suggested that the first NO production was
associated with resistance. Exogenous NO improved the
activity of phenylalanine ammonia-lyase and induced a
resistant response of Lovrin10 to the virulent strain
CY29-1, thereby providing further evidence that the first
peak of NO production was associated with resistance.
These results indicate that the first NO burst in the
immune system plays an important role in the resistant
reaction of wheat to strip rust.
Nitric oxide (NO) is an inorganic free radical that acts as a
signalling molecule with multiple biological functions in
plants. Recent studies have highlighted the important roles
of NO in disease. NO plays a key role in the activation of
pathogen-related proteins in animal and plants (Delledonne et al. 1998; Durner, Wendohenne & Klessing 1998).
It has been reported to protect chlorophyll levels in potato
leaves infected by the pathogen Phytophthora infestand
(Laxalt, Beligni & Lamattina 1997). In Leguminosae,
tobacco mosaic virus and fungi were reported to induce
phenylalanine ammonia-lyase (PAL) activity followed by
the induction of antimicrobial phytoalexins (Jones 1984).
Treatment of potato tuber tissues with the NO donor NOR18 induced an accumulation of the phytoalexin rishitin, an
endogenous antiobiotic compound (Noritake, Kawakita &
Doke 1996).
In the present study we investigated the effect of NO on
the activity of PAL and disease development in a wheat–
stripe rust system. The stripe rust (or yellow rust) disease
caused by Puccinia striiformis Westend is a serious disease
of wheat (Triticum aestivum L.) in many areas of the world.
Three epidemics of this disease occurred in China in 1950,
1964 and 1990, causing losses of wheat production of about
60, 32 and 25 million kilograms, respectively. Thus it is
important to find the mechanisms of the disease and a
method to prevent it. Due to the penetration into the stoma
by parasites, it is difficult to study the relationship between
NO production and the disease. Furthermore, the resistant
reaction of host to pathogen in plants is not the same as in
cultured cell systems.
Key-words: electron spin resonance; Lovrin 10; phenylalanine ammonia-lyase; pathogenesis-related protein; yellow
rust.
Abbreviations:
CPTIO,
2-4-carboxyphenyl-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide; CY, China yellow
rust; ESR, electron spin resonance; NO, nitric oxide; PAL,
phenylalanine ammonia-lyase; PR protein, pathogenesisrelated protein; SNP, sodium nitroprusside.
Correspondence: Professor Baolu Zhao. Fax: 8610 64871293: email: [email protected] and Academician Zhenqi Li. Fax:
8629 7092234; e-mail: [email protected]
*These authors contributed equally to this work.
© 2004 Blackwell Publishing Ltd
MATERIALS AND METHODS
Chemicals
HEPES
[N-2-hydroxyethyl-piperazine-N¢-(2-ethanesulfonic acid)], sodium nitroprusside (SNP), diethyldithiocarbamate (DETC), L-phenylalanine and 2-4carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide
(CPTIO) were purchased from Sigma Chemical Co. The
other reagents purchased in China were of analytical grade.
All reagents were used as solutions in double-distilled
water.
473
474 P. Guo et al.
Plant materials, fungus
The wheat cultivar Lovrin10 and the strains CY22-2 and
CY29-1 of stripe rust were obtained from North-West Science-technology University of Agriculture and Forestry
and each strain was identified in discriminated hosts. The
phenotypes of Lovrin10 inoculated with the clones of single
spores of CY22-2 and CY29-1 exhibited immune and susceptible reactions, respectively.
The wheat seeds to be tested were sown in 10-cmdiameter pots with 15 plants per pot and cultivated in a
greenhouse. When the first leaves were fully expanded the
seedlings were inoculated with fresh spores prepared in
advance using the brush method. They were then incubated
at temperatures of 18/12 ∞C (light/dark) with 14 h of light
per day. The infection types were recorded during the
period 16–18 d after inoculation.
Monitoring the endogenous NO in the plant by
electron spin resonance
The inoculated primary leaves were excised at appropriate
times after induction. Leaves from three plants were used
for each time point and each plant was sampled only once.
The leaves were ground in 0.1 M phosphate
buffer(pH 7.4, containing 0.32 M sucrose, 10 mM HEPES,
0.1 mM ethylenediaminetetraacetic acid (EDTA), 5 mM
thioaethylenglycol) at 4 ∞C in the presence of quartz sand.
The sample was centrifuged at 13 201 g for 20 min, and the
supernatant was incubated with 6 mM FeSO4, 12 mM
DETC and 10 mM Na2S2O3. The NO signal was then measured (Zhang et al. 2001) at room temperature using a
Bruker ER200D-SRC spectrometer (Bruker Analytik
GmbH, Germany). The conditions for ESR were as follows:
X-band; 100 kHz modulation with 3.2 G amplitude; microwave power 20 mW; central magnetic field 3385 G with scan
400 G. NO detected by ESR was expressed as the relative
intensity of the signal. Accumulation of NO generated from
the homogenate preparation during incubation of homogenate with spin trapping agent was detected, but NO was not
detected before preparation of the homogenate in this condition (Zhang et al. 2001).
Assay for the effect of exogenous NO on disease
symptom
Different concentrations of the NO donor SNP (0.5 mM,
2.5 mM) were applied to the first opened leaves of wheat
and the NO scavenger CPTIO and NO donor SNP cotreatment was used as the control. The degree of stripe rust
disease was expressed as a percentage of the diseased area
in the resistant system to that in susceptible system, with
the susceptible system being expressed as 100%.
Enzyme extraction and PAL assay
After grinding 1 g fresh leaves in liquid nitrogen with a
mortar and pestle, the enzymes in the frozen powders were
extracted by adding 0.05 g polyvinylpyrrolidone and 2 mL
buffer (pH 7.4, 0.1 M phosphate, 2 mM EDTA, 4 mM dithiothreitol), and then homogenized at 4 ∞C at 10 000 g for
25 min. After centrifugation the enzyme extract was used
for determination of PAL activity by the method reported
by Heide et al. (1989) with slight modification.
RESULTS
Assessment of the role of endogenous NO in
different phenotypes
Innoculation of the wheat cultivar Lovrin10 with the P.
striiformis Westend strain CY22-2 caused an immune reaction, whereas treatment with the CY29-1 strain led to a
susceptible reaction. For the immune phenotype no spores
of stripe rust were observed at any stage from the beginning
to the end of experiment (18 d), whereas for the susceptible
phenotype many spore colonies could be observed on
wheat leaves by 15 d after inoculation (results not shown).
The NO generated in the Lovrin10 leaves were measured
using the ESR spin trapping technique and typical spectra
showing three peaks were obtained from the leaves of
Lovrin 10 inoculated with CY22-2 or CY29-1, respectively
(Fig. 1). The spectra show that they both have a triplet
signal with aN = 12.5 G at g = 2.035. The peak at g = 2.02 is
the peak of the Cu2+ complex (Zhou et al. 1999).
The time courses of endogenous NO production in the
immune and the susceptible systems were different (Fig. 2).
Two peaks were observed during the time course of the
immune system. A rapid and strong NO production in
leaves reached a peak at 24 h after inoculation with CY222 in the early infection stage. This was followed by decreasing to a lower level after 72 h. Then it increased and reached
a second peak by 96 h in the latent development stage, and
subsequently decreased again to a lower level. However in
the susceptible system, only a single NO peak in the latent
development period was observed. The figure shows that
the phenotype is associated with the first NO burst of the
host in the early stages of infection by the pathogen, but
the second NO burst of the latent development period is
not observed.
Effect of exogenous NO on disease symptom
As NO appears to be a key factor associated with disease
symptoms, it was of interest to test the effect of exogenous
NO. Treatment of leaves of Lovrin10 with the avirulent
strain CY22-2 did not produce spore colonies on the surface of the leaves, so no symptoms of disease were observed
and multiplication of the fungi was inhibited. By contrast,
inoculation with the virulent CY29-1 strain caused many
spore colonies to be produced on the leaf surface and disease symptoms were observed. Treatment with SNP before
inoculating the virulent strain CY29-1 prevented fungal
development in a concentration-dependent manner. However co-treatment of SNP with the NO scavenger CPTIO
before inoculation of virulent CY29-1 did not block the
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 473–477
NO in the wheat-stripe rust system 475
Figure 2. The time course of endogenous NO production in the
leaves of Lovrin 10 inoculated with the strain of stripe rust, CY222 (dash-dot) and CY29-1 (solid-line), respectively. Each time point
represents the mean ± SD of three experiments (n = 3).
ated in the leaves of wheat. (a) Spectrum of (DETC)2-Fe2+-NO
complex extracted from the wheat leaves inoculated with CY22-2.
(b) Spectrum of that extracted from the wheat leaves inoculated
with CY29-1. The three peaks at g = 2.035 with aN = 12.5 G came
from the hyperfine of NO complex, the peak at g = 2.02 came from
the complex of Cu2+-complex. (c) Spectrum of the sample without
addition of FeDETC.
development of disease symptoms and had no effect on the
multiplication of strip rust (Figs 3 & 4). A control experiment revealed that SNP could not affect the germination
of stripe rust.
Area of disease
(relative value)
Figure 1. The ESR spectra of (DETC)2-Fe2+-NO complex gener-
(P < 0.05). There was no statistically significant difference
in PAL activities between the immune and susceptible systems in latent development period 72 h after inoculation
with stripe rust, the activity of PAL remained basically
constant during the latent developing period.
PAL activity had a similar time course with endogenous
NO production but the first peak of PAL activity was
delayed about 24 h after the first NO peak, suggesting that
endogenous NO may be one of the inducers of PAL activity
in the host inoculated with the avirulent strain CY22-2.
P
2.
0m
M
SN
P
SN
P
M
SN
5m
1.
1.
0m
M
M
SN
O
5m
0.
CP
TI
-1
P+
29
SN
CY
In order to demonstrate the correlation of endogenous NO
with pathogenesis-related protein in the immune and susceptible system, the time course of PAL in wheat leaves of
the two systems was determined. Inoculating leaves with
stripe rust resulted in the expression of enhanced PAL
activity within 144 h (Fig. 5). PAL was induced to a higher
level in the host treated with the avirulent strain CY22-2
than that treated with the virulent strain CY29-1. The activity of PAL was induced to the highest level 48 h after treatment with the avirulent strain CY22-2. This was 1.6-fold
higher than the level attained with the virulent strain CY291 at the same time; the difference was very distinct
P
Effect of endogenous NO on the activities of PAL
Concentration
Figure 3. The relative area of diseased leaf tissue of Lovrin10
inoculated with CY29-1 24 h after treatment with different concentrations of SNP. The value of the susceptible system is expressed
as 100%. Values represent the mean ± SD of three repeats (n = 3).
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 473–477
476 P. Guo et al.
DISCUSSION
In order to test whether exogenous NO induces the activity of PAL in leaves, we examined the effects of the exogenous NO donor SNP and an inhibitor of NO on PAL
activity. Treatment of the leaves with 0.5 and 2.0 mM NO
donor SNP generated a steady-state NO concentration of
about 2 and 4.5 mM, respectively, as measured by the haemoglobin assay (Murphy & Noack 1994). This induced a
similar high activity of PAL in the leaves to that seen after
treatment with the avirulent strain (Fig. 6). However,
cotreatment of NO scavenger CTPIO and NO donor SNP
did not improve PAL activity. This result indicates that
either exogenous NO or endogenous NO could activate
PAL.
Free radicals in dicots have been studied in detail, but not
in monocots. In this study, we investigated the time course
of endogenous NO and pathogenesis-related protein in
intact wheat leaves inoculated with different stripe rust
strains. Remarkable differences were revealed in the
dynamic changes of endogenous NO in the same cultivar
inoculated with an avirulent or a virulent strain. Furthermore NO production after early infection by pathogen
improved the activity of PAL. It was demonstrated that the
kinetics and relative accumulation of NO production was
the key factor associating the host reaction phenotype and
pathogenesis-related protein.
As NO is a free radical, there are few reliable and specific
tests owing to its rapid interconversion to other species and
chemical tests tend to respond to other nitrosyl species.
ESR provides a means to detect NO species in biological
samples. Complexes of ions such as NO(DETC)2Fe2+ are
ESR-detectable states allowing the monitoring of the time
course of endogenous NO in host leaves inoculated with
virulent or avirulent strains. The phenotype is very different
when the same cultivar Lovrin 10 is treated with differenct
strains of P. striiformis, in which endogenous NO is
detected by ESR. The two peaks of NO in the time course
of disease progression form the most significant characteristic distinguishing the immune system from the susceptible
system. Whereas in the susceptible system, NO accumulation was not observed during the early infection stage. NO
accumulation during the latent development period was
almost the same in both resistant and susceptible systems.
The time course indicated that signal differences of endogenous NO in early infection were significant when comparing the resistant and susceptible systems. Thus, the NO
Figure 5. Time course of PAL activity in the leaves from the
Lovrin10 inoculated with the strains of stripe rust CY22-2 (line)
and CY29-1(dot), respectively. Every time point represents the
mean ± SD of three experiments (n = 3), there are significant difference at key time point (P < 0.05).
Figure 6. The activity of pathogenesis-related protein PAL in
wheat leaves inoculated with avirulent strain CY22-2 or virulent
strain CY29-1, respectively. Leaves were first treated with different
concentrations of SNP then inoculated with CY29-1. Values represent the mean ± SD of three repeats (n = 3).
Figure 4. Disease symptoms observed on the leaves of Lovrin10
inoculated with virulent strain CY29-1 24 h after SNP treatment.
ck means the control Lovrin10 inoculated with virulent strain
CY29-1 24 h after treatment with SNP + CPTIO.
-1
CY
29
-2
22
0.
5m
M
CY
SN
P+
CP
TI
O
P
SN
M
0m
2.
0.
5m
M
SN
P
PAL activity
(relative units)
Effect of exogenous NO on PAL activity
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 473–477
NO in the wheat-stripe rust system 477
signal during this period may be a key factor associated
with resistance or with disease symptoms. Although the NO
signal in the latent period may be insignificant for disease
symptoms, overall, the results suggest that NO plays an
important role during interaction of host and pathogen.
Delledonne et al. (1998) found that a rapid, relatively weak
NO peak was induced by both avirulent and virulent strains
in soybean cell system suspension, but the stronger NO
peak was induced only by the avirulent strain.
Treatment of potato tuber tissues with the NO donor
NOR-18 led to an accumulation of the phytoalexin rishitin,
an endogenous antibiotic compound (Noritake et al. 1996).
However, NO did not affect P. infestans growth or infection;
its protective effect in potato is possibly related with plant
defence mechanisms (Laxalt et al. 1997). It is well known
that the infection of a tissue by a avirulent pathogen will
lead to the induction of pathogenesis-related protein
(Linthorst 1991). The results of our study have provided
insights into a possible mechanism as to how avirulent
strains induce resistance. The NO observed in early infection by pathogen was associated with improved PAL activity in the resistant system, which reached its highest activity
within 48 h. Although the activity of PAL in the susceptible
system also increased in the same period, it was significantly
lower than that seen in the resistant system. Treatment of
leaves with SNP also showed that NO induced PAL activity
and the level of activity was dependent on the NO concentration. Apparently, a threshold level of PAL activition is
necessary for the resistant expression of wheat to stripe
rust.
The phenylpropanoid pathway is considered to be important due to its role in the synthesis of a large range of
natural products in plants, including lignans, lignin, flavonoids and anthocyanins (Murphy & Noack 1994). The
first step in the phenylpropanoid biosynthetic pathway is
catalysed by phenylalanine ammonia-lyase (PAL, EC
4.3.1.5), which converts L-phenylalanine to trans-cinnamic
acid (Verpoorte 2000). In the Leguminosae, tobacco mosaic
virus and fungi were reported to induce PAL activity followed by induction of antimicrobial phytoalexins (Jones
1984).
Treatment of wheat leaves with the NO donor SNP
before inoculation the virulent strain CY29-1 induced the
resistance of Lovrin 10 to CY29-1, but cotreatment SNP
with NO scaveager CPTIO did not induce resistance. The
fact that treating the fungi with SNP did not inhibit its
germination suggests the contribution of SNP to the resistance was NO inducing a resistant reaction in the host
rather than NO and other SNP derivates directly killing the
pathogen. This is supported by evidence that the host was
still susceptible when the wheat leaves were treated with
the virulent strain CY29-1 and SNP simultaneously, and the
exogenous NO induced the imcreasing activity of PAL. The
induced resistance was dependent on the exogenous NO
concentration, although the resistant reaction did not reach
the highest immune level. It is concluded that the defence
reponse induced by NO was necessary but not sufficient for
the immune reaction.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (30070196) and the Ph.D. Foundation
of China (97007003).
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Received 29 September 2003; received in revised form 1 December
2003; accepted for publication 3 December 2003
© 2004 Blackwell Publishing Ltd, Plant, Cell and Environment, 27, 473–477