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SHORT COMMUNICATION
DEFENCE GENES AND ANTIOXIDANT ENZYMES IN STAGE-DEPENDENT
RESISTANCE TO RICE NECK BLAST
Z.N. Hao, L.P. Wang and R.X. Tao
Institute of Plant Protection and Microbiology, Zhejiang Academy
of Agricultural Sciences, Hangzhou 310021, China
SUMMARY
The rice variety Jiajing3768 is susceptible to neck
blast at the booting stage (BS) but resistant at the full
heading stage (FHS). This variety was used to analyze
the inducible expression of defence genes and activities
of antioxidant enzymes in the necks, at both the BS and
FHS, after inoculation with Magnaporthe oryzae. We
found that defence genes PR1b (pathogenesis-related
class 1b), PBZ1 (probenazole-inducible gene), PAL
(phenylalanine ammonia-lyase), and CHS (chalcone synthase) may play roles in the resistance difference to neck
blast between the BS and FHS in Jiajing3768. No significant differences were observed between the BS and
FHS in the inducible expression of PR1a, PR4, and
JIOsPR10 (jasmonic acid induced PR 10). The antioxidant enzymes superoxide dismutase, peroxidase, catalase, glutathione reductase, and malondialdehyde coordinately participated in the stage-dependent resistance
difference in Jiajing3768, and that oxidative damage is
lower in the necks at the FHS than at the BS.
Key words: Oryza sativa, Magnaporthe oryzae, panicle
stage, stage resistance, defence response.
Rice blast, caused by Magnaporthe oryzae, is the most
devastating fungal disease of rice frops (Couch and
Kohn, 2002). It has two forms, leaf blast and neck (or
panicle) blast, the latter is the major cause of yield losses. In the field, there are three crucial stages that control
neck blast: the booting stage (BS), the preliminary heading stage (PHS), and the full heading stage (FHS). Our
unpublished work indicates that resistance to neck blast
at the BS positively correlates with resistance to neck
blast at the PHS or the FHS in many rice varieties. Nevertheless, a few varieties exhibit significantly higher or
lower resistance to neck blast at one panicle stage. This
Corresponding author: R.X. Tao
Fax: +86.0571.86404226
E-mail: [email protected]
form of resistance can be referred to as stage-dependent
resistance the mechanism underlying it, however, remain largely unknown. Studies on rice resistance to
neck blast mainly focus on the PHS (Pattama et al.,
2002; Tanee et al., 2008), which is insufficient for investigating rice resistance in some varieties that exhibit
stage-dependent resistance to this disease.
Plants defend themselves against pathogen challenges by the activation of defence response pathways
(Staskawicz et al., 1997). The recognition of pathogen
avirulence gene products by plant resistance gene products leads to the rapid, coordinated expression of defence genes, whose products participate in fighting
pathogen infection (Leo and Maarten, 2000). The
speeds of activation and the expression levels of defence
genes vary in different plant-pathogen interactions.
Plants have also developed complex antioxidant defence systems that respond to biotic and abiotic stresses
and mitigate the deleterious effects of reactive oxygen
species (ROS) (Panda, 2007). The levels of ROS and the
extent of oxidative damage depend largely upon the level of coordination among ROS-scavenging enzymes
(Liang et al., 2003). In this report, we examined the inducible expression of some known defence genes, the
activities of some antioxidant enzymes, and malondialdehyde (MDA) levels in the necks at both the BS and
FHS.
The rice variety Jiajing3768 (Oryza sativa subsp.
japonica) is susceptible to neck blast at the BS but resistant at the FHS. Rice plants were grown under natural
light in a greenhouse (20-30°C) for inoculation experiments. A 1×105 conidia ml-1 conidial suspension of the
M. oryzae strain 06-257 (race ZG1) was obtained according to Campbell and Ronald (2005).
The emerging panicles were collected at the BS and
FHS (Rao et al., 2005). Neck pieces 6 cm long, containing two or three nodes, were cut and placed onto the
paper filters in glass dishes (10 cm diameter). The neck
nodes were brushed with the conidial suspension and
were incubated in a moist chamber at 26°C. The necks
were collected at 1, 2, 3, 4, and 5 days post inoculation
(dpi), frozen in liquid nitrogen and stored at -80oC.
Necks treated with sterile water for 1, 2, 3, 4, and 5 days
were used as controls.
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Table 1. Primers and probes used for quantitative RT-PCR.
Gene name
PR1a
Accessiona
AJ278436
PR1b
U89895
PR4
AY050642
Sequence
Fb:5’-GGTGTCGGAGAAGCAGTGGTA-3’
Rc:5’-GCGAGTAGTTGCAGGTGATGAAG-3’
Pd:5’-CCGGAGGGGAGCTCGTGCG-3’
Fb:5’-AGGCGTTCGCGGAGAACTA-3’
Rc:5’-GAAGAGGTTCTCGCCAAGGTT-3’
Pd:5’-CAGCCAGAGGAGCGGCGACTG-3’
Fb:5’-CATTATTACAACCCACAACAGAACAA-3’
Producte (bp)
170
92
71
Rc:5’-GCATCCCATGTGGCACAAT-3’
Pd:5’-TGGGACCTGAACAAAGTGAGCGCA-3’
JIOsPR10
AF395880
Fb:5’-GCAGCGTCAGGCAGTTCAA-3’
68
Rc:5’-GAACTCCAGCCTCTCCTTCATG-3’
Pd:5’-TTCACCTCAGCCATGCCATTCAGC-3’
PBZ1
D38170
Fb: 5’-ATGGCTACTATGGCATGCTCAA-3’
78
Rc: 5’-CCTCTTCATCTTAGGCGTATTCG-3’
Pd: 5’-AGGACTACCTCGTCGCTCACCCT-3’
PAL
X16099
Fb:5’-GGTGTTCTGCGAGGTGATGA-3’
73
Rc:5’-AGGGTGGTGCTTCAGCTTGT-3’
Pd:5’-CAAGCCGGAGTACACCGACCACC-3’
CHS
AB000801
Fb:5’-CCGGCGAACTGCGTGTAC-3’
101
Rc:5’- CACATCCTCTTGAACTTCTCCTTGA-3’
Pd:5’-TCAGGATCACCAAGAGCGAGCACATG-3’
Actin
X15865
Fb:5’-GAGCTACGAGCTTCCTGATGGA-3’
65
Rc:5’-CCTCAGGGCAGCGGAAA-3’
Pd:5’-AGGTTATCACCATTGGTGCTGAGC-3’
a
GenBank Accession numbers of genes; b Forward primers used for quantitative RT-PCR;
c
Reverse primers used for quantitative RT-PCR; d TaqMan probes used for quantitative RT-PCR;
e
Amplification product sizes of quantitative RT-PCR.
The inducible expression of defence genes was analyzed by quantitative RT-PCR according to the manufacturer’s instructions (ShineGene, China), using an
ABI PRISM 7000 Sequence Detection System (Applied
Biosystems, USA). TaqMan primers and probes were
designed using the Primer Premier 3.0 software (Ap-
plied Biosystems, USA). according to gene sequences in
the GenBank database (Table 1). PCR was carried out
at 93°C for 2 min, followed by 40 cycles at 93°C for 5
sec (denaturation), and annealing at 60°C for 30 sec.
The actin gene was used as the house-keeping gene.
Superoxide dismutase (SOD; EC 1.15.1.1), peroxi-
Fig. 1. A. Necks of cv. Jiajing3768 at the booting stage, 10 days post-inoculation with Magnaporthe
oryzae strain 06-257 (ZG1). Most of the necks display typical blast lesions with production of conidia,
and etiolation. B. Necks of cv. Jiajing3768 at the full heading stage showing resistant-type lesions.
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dase (POD; EC 1.11.1.7), catalase (CAT; EC 1.11.1.6),
glutathione reductase (GR; EC 1.6.4.2), and MDA levels were measured with kits according to the manufacturer’s instructions (JianCheng, China).
Jiajing3768 showed significant differences between
the BS and FHS in resistance to neck blast after inoculation with M. oryzae (Fig. 1). The inoculated necks
were checked at 10 DPI, and the disease incidence, lesion length, and number of conidia in the necks at the
BS were 97.1%, 48.5 mm, and 27530 conidia ml-1, respectively. The corresponding indexes at the FHS were
25.6%, 2.4 mm and 125 conidia ml-1, respectively. We
also confirmed that Jiajing3768 was susceptible to neck
blast at the PHS (data not shown).
The expression levels of PR1a, JIOsPR10, PBZ1 and
PAL were all significantly lower in the uninoculated
necks at the BS than at the FHS (Fig. 2). At both stages,
the PAL transcript showed the highest levels. The lowest levels were seen for the PR1a transcript at the BS,
and the PR1b transcript at the FHS.
The induction of defence gene expression in the
necks at the BS and FHS was analyzed at 1-5 dpi (Fig.
3). The PR1a transcript level increased until the 5th dpi
at the BS, by only 1.3-fold compared with the control.
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Fig. 2. The expression of defence genes in the uninoculated
necks at the booting stage (BS) and full heading stage (FHS)
analyzed by quantitative RT-PCR. Transcript levels of defence
genes were calculated relatively to the actin gene. Error bars
represent the standard deviation of the mean from three independent experiments. Asterisks indicate a significant difference (P<0.05, t-test) in the necks between the booting and
full heading stage within the same defence gene.
Although PR1a expression was up-regulated throughout the experimental period at the FHS, no significant
differences were observed between the BS and FHS except at 3 dpi. OsPR1a, a rice acidic PR class 1 protein,
is highly responsive to pathogen attack, wounding, and
Fig. 3. Inducible expression of defence genes in the necks at the booting (BS) and full heading stage (FHS) in cv. Jiajing3768 after
infection with the Magnaporthe oryzae strain 06-257 (ZG1) analyzed by quantitative RT-PCR. The necks were examined at 1, 2, 3,
4 and 5 days post-inoculation. Transcript levels of defence genes were normalized to those of the house-keeping gene actin. Expression levels of defence genes in inoculated samples were calculated relatively to those of controls treated with sterile water. Error bars represent the standard deviation of the mean from three independent experiments. Circles indicate a significant difference (P<0.05, t-test) between controls and inoculated samples. Asterisks indicate a significant difference (P<0.05) in the necks between booting and full heading stage at the same treatment time.
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Table 2. Activities of antioxidant enzymes and MDA levels in the uninoculated necks at the booting stage and full
heading stage in Jiajing3768.
Development stage
SOD (U mg-1
protein)
POD (U mg-1
protein min-1)
CAT (U mg-1
protein min-1)
GR (U mg-1
protein min-1)
MDA (nmol mg-1
protein)
Booting stage
Full heading stage
78.74 ± 3.18
60.69 ± 11.36
58.16 ± 3.40
64.71 ± 2.35
1.29 ± 0.62
5.21 ± 1.00*
17.13 ± 4.41
21.22 ± 2.44
1.82 ± 0.17
2.90 ± 0.32*
Values represent the mean from three independent experiments ± standard deviation; * values represent a significant
difference in the necks between the booting stage and full heading stage according to the Student’s t-test with P<0.05.
salicylic acid. The blast fungus infection can induce
PR1a transcript accumulation in both compatible and
incompatible interactions (Agrawal et al., 2000a). However, our data suggest that PR1a may not be involved in
the resistance difference to neck blast in Jiajing3768 between the BS and FHS.
PR1b expression was up-regulated at both the BS
and FHS during the entire duration of the experiment,
except at 3 dpi at the BS. Interestingly, the inducible expression levels of PR1b at 3, 4 and 5 dpi compared to
the level in control were significantly higher at the FHS
than at the BS, indicating that this gene may play an important role in stage-dependent resistance to neck blast
in Jiajing3768. The rice PR1b gene, which encodes a ba-
sic PR protein, is commonly used as a marker for systemic acquired resistance. The deduced amino acid sequence of OsPR1b has only 63.1% homology with the
OsPR1a protein (Agrawal et al., 2000b), which may explain why PR1a and PR1b showed different expression
patterns in this study.
OsPR4 was cloned from a differentially expressed
cDNA library of rice leaves infected with the blast
pathogen (Agrawal et al., 2003). In this report, PR4 expression was induced throughout most of the experimental period at both the BS and FHS, except at 1 dpi
at the FHS. PR4 expression was highest at 2 dpi (2.1fold) at the BS and at 4 dpi (2.3-fold) at the FHS, compared with the controls, respectively. This suggests that
Fig. 4. SOD (a), POD (b), CAT (c) and GR (d) activities, and MDA levels (e) in the necks at the booting stage (BS) and full heading stage (FHS) in cv. Jiajing3768 after inoculation with Magnaporthe oryzae (DPI = days post-inoculation). Activities and levels
are expressed relatively to controls (=100%, dashed line) at the respective times. Error bars represent the standard deviation of
the mean from three independent experiments. Circles indicate a significant difference (P<0.05, t-test) between controls and inoculated samples. Asterisks indicate a significant difference (P<0.05) in the necks between the booting and full heading stage at the
same treatment time.
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PR4 may not be involved in the resistance difference between the BS and FHS to neck blast in Jiajing3768.
JIOsPR10, induced by treatment with jasmonic acid,
salicylic acid, and pathogens, was discovered among five
rice PR-10 genes (Kim et al., 2008). Our results suggest
that JIOsPR10 had no effect on stage-dependent resistance to neck blast in Jiajing3768.
PBZ1 is induced by probenazole, and thus is also
called OsPR10a (Liu and Ekramoddoullah, 2006). PBZ1
expression was up-regulated at both the BS and FHS
throughout most of the experimental period, except at 1
dpi at the FHS. Furthermore, the inducible expression
levels of PBZ1 at 4 and 5 dpi compared with the controls were higher at the FHS than at the BS, suggesting
that PBZ1 could have an important function in the resistance difference to neck blast between the BS and
FHS in Jiajing3768. Rapid induction and high levels of
defence gene expression are necessary for plants to fight
pathogens. In most cases, the inducible levels of gene
expression in compatible interactions are lower than in
incompatible interactions.
No significant induction of PAL expression was observed at the BS, but induction was significant at 4 dpi
(3.6-fold) and 5 dpi (2.6-fold) at the FHS. However,
CHS expression was down-regulated at the BS but upregulated at the FHS throughout the experimental period. Therefore, we suggest that PAL and CHS could play
important roles in stage-dependent resistance to neck
blast in Jiajing3768. PAL is the first enzyme in the
phenylpropanoid pathway, which has important functions in plants following exposure to environmental
stresses and pathogen attack (Minami et al., 1989). CHS
is the first enzyme in flavonoid and isoflavonoid biosynthesis, which is part of the phenylpropanoid pathway
(Zabala et al., 2006), which may explain why PAL and
CHS showed similar patterns of inducible expression after infection with M. oryzae at both growth stages.
CAT activity and MDA levels in the uninoculated
necks were significantly higher at the FHS than at the
BS (Table 2). SOD activity was lower at the FHS than at
the BS. POD and GR had similar activities at both
growth stages.
All antioxidant enzymes, except for SOD, showed
different responses to M. oryzae in the necks at between
the BS and FHS (Fig. 4). In active oxygen-scavenging
systems, superoxide radicals generated in plants are
converted to H2O2 by the action of SOD (Bowler et al.,
1992). However, our data imply that SOD minimally
participates in the resistance difference to neck blast between the BS and FHS in Jiajing3768 (Fig. 4 a).
POD is one of the most important enzymes active in
elimination of ROS and catalyzes the oxidoreduction of
various substrates using hydrogen peroxide. Many reports have suggested that POD plays a role in resistance
to pathogens (Kawaoka et al., 2003; Caruso et al., 2001).
POD activities increased slightly over controls at the
Hao et al.
751
FHS, but decreased at the BS (Fig. 4 b), suggesting that
POD could be important in stage-dependent resistance
to neck blast in Jiajing3768. POD and CAT, another
major ROS-scavenging enzyme, showed similar trends
(Fig. 4 c), thus these two enzymes might more efficiently
clear ROS at the FHS than at the BS.
GR catalyzes the reduction of oxidized glutathione to
reduced glutathione (GSH) with the accompanying oxidation of NADPH, and this reaction is important to the
ascorbate (AsA)-GSH cycle, the main mechanism for the
detoxification of ROS in plants (Noctor and Foyer, 1998).
However, few studies have been done to analyze the functions of GR in plant-pathogen interactions. Our data indicate that Jiajing3768 exhibits similar changes in GR activity as POD, at both stages (Fig. 4 d), hinting that it may
also play an important role in the resistance difference between the BS and FHS to neck blast in Jiajing3768.
Active oxygen radicals may induce chain-like peroxidation of unsaturated fatty acids in the membranes, leading to the formation of lipid peroxidation products such
as malondialdehyde (MDA) (Mishra et al., 2008). However, no significant changes in MDA levels were observed in either stage, except that MDA was significantly
higher at 2 dpi at the FHS than at the BS (Fig. 4 e).
Therefore, we propose that POD, CAT and GR participate to different degrees in stage-dependent resistance
to neck blast in Jiajing3768. As a result, in Jiajing3768
oxidative damage is lower in the necks at the FHS than
at the BS after infection by M. oryzae.
ACKNOWLEDGEMENTS
This work was supported by National Foundation
for Natural Science (China, No. 30600412) and Zhejiang Province Key Project for Science and Technology
(China, No. 2006C12101).
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