Plant Cell Physiol. 43(7): 823–831 (2002)
JSPP © 2002
Chloroisonicotinamide Derivative Induces a Broad Range of Disease Resistance in Rice and Tobacco
Hideo Nakashita 1, 2, 9, Michiko Yasuda 1, 3, 4, Masanori Nishioka 1, 5, Satoru Hasegawa 1, 6, Yuko Arai 1, 4, 7,
Masakazu Uramoto 6, Shigeo Yoshida 2, 3 and Isamu Yamaguchi 1, 4, 8
1
Microbial Toxicology Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198 Japan
Plant Function Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198 Japan
3
Laboratory for Growth Regulation, Plant Science Center, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198 Japan
4
Graduate School of Science and Engineering, Saitama University, 255 Shimookubo, Saitama-shi, Saitama, 338-8570 Japan
5
Biological Research Laboratories, Nissan Chemical Industries, Ltd., 1470 Shiraoka, Minamisaitama, Saitama, 349-0294 Japan
6
Department of Applied Biological Chemistry, Tamagawa University, Machida, Tokyo, 194-8610 Japan
7
Present address: Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198 Japan
8
Present address: Laboratory for Remedition Research, Plant Science Center, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama, 351-0198 Japan
2
;
and a source of serious stress for plants, which have evolved
methods to protect themselves with a unique self-protection
system as well as various morphological adaptations. The primary response in this self-protection system involves specific
pathogen recognition and a rapid induction of localized host
cell death (Ross 1961). The secondary response consists of
induced resistance to protect the plant’s body from further
pathogenic attacks (McIntyre et al. 1981, Kuc 1982).
These responses are governed by hormonal regulation, in
which salicylic acid (SA), jasmonic acid (JA) and ethylene can
each contribute. Plants can also activate distinct defense
signaling pathways, depending on the type of invading pathogen (Dong 1998, Glazebrook 1999, Maleck and Dietrich 1999,
Pieterse and Van Loon 1999). Systemic acquired resistance
(SAR) is activated after infection by a necrotizing pathogen
and confers resistance against a broad spectrum of pathogens in
uninfected parts of the plant (Chester 1933, Durner et al. 1997).
The existence of SAR has been demonstrated in many plant
species, while SA was specifically identified as a signaling
molecule during SAR development in dicotyledonous plants,
such as tobacco and Arabidopsis (Gaffney et al. 1993, Delaney
et al. 1994). Non-pathogenic rhizosphere-colonizing Pseudomonas bacteria, on the other hand, trigger a similar systemic
resistance (called rhizobacteria-mediated induced systemic
resistance, or ISR) that utilizes JA-mediated but not SAmediated signaling (Knoester et al. 1999, Pieterse et al. 2000,
van Wees et al. 1999).
Detailed study of the SAR mechanism has resulted in the
identification of several chemicals capable of activating SAR
and several mechanisms of resistance induction. In addition to
these SAR activators, b-aminobutyric acid has been reported to
induce disease resistance in Arabidopsis by activating a signaling pathway distinct from SAR and ISR (Zimmerli et al. 2000).
Thus, identifying chemicals capable of inducing disease resistance would be useful not only for investigating known induced
resistance mechanisms, but also for identifying novel ones.
Although many types of compounds have been reported to
Systemic acquired resistance (SAR) is a potent innate
immunity system in plants that is effective against a broad
range of pathogens. SAR in dicotyledonous plants such as
tobacco and Arabidopsis has been partially elucidated and
is mediated by salicylic acid (SA). However, the SAR mechanism of monocotyledonous rice plants remains to be clarified, although some similarities between SAR mechanisms
in both types have been reported. Here we have characterized N-cyanomethyl-2-chloroisonicotinamide (NCI) as an
effective SAR inducer in both plant species. Soil drench
application of NCI induces a broad range of disease resistance in tobacco and rice and, more specifically, PR gene
expression in tobacco. Both SA measurements in wild-type
NCI-treated tobacco and pathogenic infection studies using
NahG transgenic tobacco plants indicate that NCI-induced
resistance enhancement does not require SA. Therefore, it
is suggested that NCI induces SAR by triggering signaling
at the same level as or downstream of SA accumulation as
do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl
ester and 2,6-dichloroisonicotinic acid. The fact that all of
these chemicals are effective in rice and tobacco suggests
that several common components function in disease resistance in both plant species.
Keywords: Systemic acquired resistance — Tobacco — Rice
— Magnaporthe grisea — Pseudomonas syringae.
Abbreviations: BIT, benzisothiazole; BTH, benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester; INA, 2,6-dichloroisonicotinic
acid; ISR, rhizobacteria-mediated induced systemic resistance; JA, jasmonic acid; NCI, N-cyanomethyl-2-chloroisonicotinamide; PBZ,
probenazole; Pst, Pseudomonas syringae pv. tabaci; SA, salicylic acid;
SAR, systemic acquired resistance; TMV, tobacco mosaic virus.
Introduction
Diseases caused by microorganisms are an inevitable fact
9
Corresponding author: E-mail, [email protected]; Fax, +81-48-462-4959.
823
824
Chemically induced SAR in rice and tobacco
Fig. 1 Chemically induced disease resistance in rice. (A) Structures of chemicals capable of inducing disease resistance. NCI, N-cyanomethyl-2chloroisonicotinamide; BTH, benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester; BIT, benzisothiazole; SA, salicylic acid. (B) NCIinduced resistance against rice blast disease. Plants were treated with 0.5 or 5 mg pot–1 NCI by soil drenching 5 d prior to challenge inoculation
with M. grisea. SAR activator BTH (0.5 or 5 mg pot–1) was used as positive control. Each experiment was performed with 3–6 pots, each containing seven plants. Infection on leaf-4 was calculated 5 d post inoculation. Values are shown as the means ± SD. The experiment was repeated twice
with similar results. (C) NCI-induced resistance against rice bacterial blight disease. Plants were treated with NCI (0.05, 0.5 or 5 mg pot–1), BTH
(0.05, 0.5 or 5 mg pot–1) and BIT (0.5 or 5 mg pot–1) using the soil drench method 5 d prior to challenge inoculation with X. oryzae pv. oryzae.
Each experiment contains 20–40 plants, and the length of the bleached part of infected leaves was measured 12 d after challenge inoculation.
Means ± SD are presented. NT, not tested. Statistical analysis (AVOVA) indicates significant differences between the control and chemical-treated
groups (P<0.01).
induce disease resistance in plants, a detailed induction mechanism has only been delineated for a few compounds. SAR,
induced via an SA-mediated signaling pathway, is the bestcharacterized mechanism of induced resistance. However, only
three classes of inducers have been identified: benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) (Friedrich et
al. 1996, Görlach et al. 1996, Lawton et al. 1996) and 2,6dichloroisonicotinic acid (INA) (Métraux et al. 1990, Uknes et
al. 1992), which induce SAR without SA accumulation, and
probenazole (PBZ) and its derivative benzisothiazole (BIT)
(Midoh and Iwata 1997, Watanabe et al. 1979, Yoshioka et al.
2001), which induce SAR through SA biosynthesis (Fig. 1A).
These chemicals exhibit several essential criteria of SAR
inducers: they induce a broad range of disease resistance; their
effects are not due to their antibiotic activities; and they induce
an SAR molecular marker, PR gene expression, in plants.
Despite extensive studies with these chemicals over years,
many parts of the signaling pathway still remain to be clarified; for example, no targets of these chemicals have been
determined thus far.
In contrast to the case with dicotyledonous plants, the
requirement of SA for the SAR development in monocotyledonous rice plants has been disputed (Silverman et al. 1995,
Midoh and Iwata 1997). There is no evidence to date that SA
functions in induced resistance in rice. However, NPR1, a key
component of SAR in Arabidopsis, has also been identified in
rice (Cao et al. 1994, Delaney et al. 1995, Shah et al. 1997,
Chern et al. 2001). In addition, SAR inducers such as BTH and
PBZ have been reported to induce disease resistance in monocotyledonous plants (Watanabe et al. 1979, Schweizer et al.
1999). These observations suggest that the defense mechanisms
of these two plant species are similar, an intriguing possibility
since few common features between these species are known.
Because of the potential importance of chemicals effective in
both plant species to resolve these questions, we have searched
and characterized several active chemicals.
N-cyanomethyl-2-chloroisonicotinamide (NCI) (Fig. 1A)
has been reported to induce resistance against rice blast disease, but remains to be tested for the ability to induce SAR or
other resistance (Yoshida et al. 1990). Several biochemical
responses in NCI-treated rice to rice blast fungus are similar to
those in PBZ-treated plants; these include enhanced respiration
and lipid metabolism, as well as accelerated activity of lipoxygenase and peroxidase (Seguchi et al. 1992). If NCI is indeed
an SAR inducer, it would be expected to stimulate the signaling
pathway in a different manner than other chemicals, due to the
Chemically induced SAR in rice and tobacco
825
Fig. 2 NCI induced disease resistance in tobacco. Tobacco plants (N. tabacum cv. Xanthi nc) were grown in a growth chamber under 16 h day/
8 h night conditions at 22°C. Five-week-old plants were used for the experiments. Plants were pretreated with water (control), 1, 3 or 6 mg pot–1
NCI, or 1 mg pot–1 BTH using the soil drench method 5 d prior to inoculation with pathogens. (A) NCI enhanced N-gene mediated resistance
against TMV. Photograph of lesion formation of TMV was taken 5 d after inoculation on control and NCI (3 mg pot –1)-treated plants. (B) Average size of TMV lesions. Lesions were measured 5 d following the TMV inoculation. Each experiment was performed with five plants and two
leaves of each were inoculated with TMV. Values are shown as the means ± SD. The experiment was repeated three times with similar results. (C)
Growth of Pseudomonas syringae pv. tobaci in tobacco leaf tissues. Each experiment was performed with three plants and two samples were prepared from each plant. Closed triangles, control; open circles, 3 mg pot–1 NCI; closed circles, 6 mg pot–1 NCI. Values are shown as the means ±
SD. The experiment was repeated three times with similar results. (D) Effect of NCI against powdery mildew fungus in tobacco. Conidia of O.
lycopersici were powdered on the leaves of the pretreated plants and cultured in a greenhouse at 22°C for 7 d. Each experiment was performed
with three plants and lesions on three leaves of each plant were measured. Values are shown as the means ± SD. Statistical analysis (AVOVA)
indicates significant differences between the control group and NCI (3 mg pot–1) or BTH treated group (P<0.01). The experiment was repeated
three times with similar results.
unique cyano-group in the NCI structure. As tobacco is a suitable plant model for the evaluation of SAR inducers, we
assessed the possibility that NCI induces disease resistance in
this plant. Here we show that NCI exhibits all the criteria of an
SAR inducer in tobacco and can induce a broad range of disease resistance.
Results
NCI induces disease resistance in rice
NCI was first reported as a synthetic chemical capable of
inducing resistance against rice blast disease, but without direct
antifungal activity at the effective concentration (Yoshida et al.
1990). NCI was also demonstrated to be effective against the
rice bacterial blight disease. These findings suggested that NCI
activates an undetermined disease resistance mechanism in
rice. In these studies, 8% NCI granule containing various other
ingredients was applied to plants via soil drench method. To
avoid the influence of chemical additives, we used pure NCI to
evaluate its disease resistance induction activity. First, we
examined whether pure NCI can induce disease resistance in
rice against pathogens.
826
Chemically induced SAR in rice and tobacco
We chose rice blast disease (caused by Magnaporthe grisea) and bacterial blight disease (caused by Xanthomonas
oryzae pv. oryzae) as test diseases, because their development
mechanisms are markedly different from each other. NCI was
applied via the soil drench method and the challenge inoculation was performed 5 d later. To assess the effects of NCI, we
used the Oryza sativa cv. Aichiasahi–M. grisea race 007
model, which upon interaction with the plant produces spreading necrotic lesions. While many spreading lesions appear on
the leaves of water-treated control plants, sparse lesions were
seen on the leaves of NCI- and BTH-treated plants. Treatment
with 0.5 mg pot–1 NCI resulted in over 80% protection from
pathogenic infection (Fig. 1B), without any visible morphological changes of leaf blades and spontaneous lesions by NCI
treatment (data not shown), confirming the effect of NCI
against rice blast disease. In the previous report, it was suggested that NCI is related to the resistant reaction of rice plants,
because the treatment of NCI, as well as with PBZ, increased
the ratio of minute brownish lesions to spreading lesions
(Yoshida et al. 1990), which was also observed in this experiment (data not shown).
Treating O. sativa cv. Aichiasahi plants with NCI (0.05,
0.5 or 5 mg pot–1) also reduced disease symptoms caused by
infection with the virulent pathogen X. oryzae pv. oryzae race
003. The effect of NCI was dose-dependent, with 5 mg pot–1
NCI resulting in about 90% protection from pathogenic infection (Fig. 1C). Thus, the NCI-induced resistance in rice plants
is effective not only against fungal disease but also against bacterial disease. It appears that the effectiveness of resistance
induction is dependent on the plant and not on the pathogen,
which supports the theory that these chemicals stimulate the
innate immunity system of rice plants. In rice, NCI and BTH
exhibited the same level of resistance induction against the
pathogens tested.
NCI induces a broad range of disease resistance in tobacco
SAR activators such as BTH, INA and BIT induce disease resistance in both monocotyledonous rice and dicotyledonous tobacco plants, although the SAR mechanism of rice has
not been elucidated. It seems reasonable that NCI would be an
SAR activator as well, since it was effective in rice against two
different kinds of pathogens. We used the tobacco plant to
assess the ability of NCI to enhance resistance to infection of
various pathogens, because tobacco is a suitable model for estimating the capability of chemicals to induce disease resistance
(Ward et al. 1991, Friedrich et al. 1996). Nicotiana tabacum cv.
Xanthi nc possesses the N gene, which confers resistance to
tobacco mosaic virus (TMV) (Whitham et al. 1994); consequently, its defense response to TMV infection results in a
necrotic lesion. SA and SAR activators enhance this resistance
and reduce the size of lesions (Ward et al. 1991, Friedrich et al.
1996, Durner et al. 1997). NCI treatment was performed using
the soil drench method, with challenge inoculation performed
5 d later. The average lesion size in NCI-treated plants was
Fig. 3 Effect of NCI on NahG transgenic tobacco. NahG transgenic
tobacco plants were grown in a growth chamber under 16 h day/8 h
night conditions at 22°C, and 5-week-old plants were used for experiments. Plants were treated with water (control), 3 mg pot–1 NCI, or
1 mg pot–1 BTH using the soil drench method 5 d prior to inoculation
with TMV (A) or O. lycopersici (B).
~50% smaller than that of water-treated control plants, indicating that NCI enhanced N gene-mediated resistance just as BTH
and BIT (Fig. 2A, B).
Next, we assessed the effect of NCI on the interaction
between tobacco plants and a virulent bacterial pathogen, Pseudomonas syringae pv. tabaci (Pst). N. tabacum cv. Xanthi nc
does not have a resistance gene specific to Pst and the relationship between this plant and Pst is compatible. Susceptibility was
estimated by measuring bacterial growth in leaf tissues after
challenge infection. Treatment with 3 or 6 mg pot–1 NCI by soil
drenching inhibited bacterial growth in the infected tissues relative to the water-treated control plants (Fig. 2C), although NCI
did not show any direct anti-microbial activity in liquid culture
at concentrations of up to 500 mg ml–1 (data not shown). This
indicates that NCI induces resistance to Pst in tobacco plants.
Treating tobacco plants with 1 or 3 mg pot–1 NCI also
reduced symptoms of disease caused by infection with the virulent fungal pathogen Oidium lycopersici (powdery mildew).
Both NCI and BTH induced resistance to the progression of
this disease, as measured by lesion size (Fig. 2D). As O. lycopersici is an obligate pathogen, the direct effect of NCI on its
growth could not be determined. Considering all of these
results, we conclude that, in tobacco, NCI induces SAR-like
disease resistance to a broad range of pathogens.
Chemically induced SAR in rice and tobacco
827
Fig. 4 Accumulation of free and total salicylic acid in wild-type
tobacco plants treated with NCI. Leaves were harvested at the indicated times after treatment with 5 mg pot–1 NCI or water, and free and
total SA (free SA + SAG) levels were quantified using HPLC. Open
circles, NCI-treated plants; closed circles, control plants.
Since foliar application of other SAR inducers (including
SA) is effective on tobacco, we also examined whether NCI
treatment via foliar spraying was able to induce disease resistance. Spraying 1 or 2.5 mM NCI onto the leaves resulted in the
induction of enhanced resistance against TMV infection just as
in soil drenching, but was ineffective against infection with Pst
and O. lycopersici (data not shown). By contrast, foliar treatment with 0.2 mM BTH was effective against all of three pathogens tested (data not shown). This suggested that NCI could
not be incorporated from tobacco leaf surface.
NCI induced resistance in tobacco without the accumulation of
SA
In tobacco, SAR development is associated with SA biosynthesis. Some SAR activators such as INA and BTH induce
SAR without SA accumulation, by stimulating pathways downstream of SA, while BIT requires SA biosynthesis to induce
SAR (Vernooij et al. 1995, Friedrich et al. 1996, Yoshioka et al.
2001). To determine whether NCI requires SA for disease
resistance induction, we examined the effect of NCI on the
NahG transgenic tobacco plant, which is unable to accumulate
SA due to the expression of salicylate hydroxylase, an SAdegrading enzyme (Gaffney et al. 1993, Delaney et al. 1994).
In the case of TMV infection, the average lesion size in NCItreated plants was smaller than that of water-treated control
plants (Fig. 3A). NCI treatment of NahG plants induced statistically significant resistance against O. lycopersici (Fig. 3B).
These results indicate that NCI does not require SA to induce
SAR-like disease resistance.
To confirm that NCI-mediated activation of the defense
response is SA-independent, the levels of free and total SA
(free SA plus salicylic acid glucoside, SAG) were measured in
NCI- and water-treated tobacco plants over a 5-day time
Fig. 5 Induction of SAR marker gene expression in tobacco by NCI.
Each lane was loaded with 5 mg total RNA. rRNA was used as an
internal control for gel loading and transfer. (A) RNA gel blot analysis
of acidic PR-1, PR-2 and PR-5 gene expression in the leaves of plants
treated with H2O or NCI. Wild-type and NahG transgenic tobacco
plants were treated with H2O or NCI (5 mg pot–1) using the soil drench
method. Leaves were collected 3 or 5 d after treatment. (B) RNA gel
blot analysis of acidic PR-1 gene expression in leaves treated with
H2O (control, C), NCI or BTH. Wild-type tobacco plants were treated
with H2O, NCI (1, 2.5 or 5 mM), or BTH (0.2 mM) via foliar spraying. Leaves were collected 5 d after treatment. (C) RNA gel blot analysis of acidic PR-1 gene expression in leaves infiltrated with H2O or
NCI. Leaves of wild-type and NahG transgenic tobacco plants were
infiltrated by H2O or NCI (0.5 or 2.5 mM). Leaves were collected 3 d
after treatment and both infiltrated areas and a further 2–5 cm area
were used for RNA extraction.
course. Soil drench application of NCI did not increase the levels of either free or total SA, confirming that NCI does not
stimulate SA biosynthesis (Fig. 4).
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Chemically induced SAR in rice and tobacco
PR gene expression associated with NCI-induced resistance
Some of PR proteins are coordinately expressed in
tobacco leaves during the induction and maintenance of SAR
and these are also expressed during SAR induced by chemical
plant activators such as SA and BTH (Ward et al. 1991). The
expression of acidic PR-1 (PR-1a), encoding one of these PR
proteins, is a useful molecular marker for SAR and its expression in NCI-treated plants was examined. The Northern blot
analysis indicated that the transcript for acidic PR-1 accumulated in leaves of tobacco treated with NCI via soil drenching
(Fig. 5A). The transcripts for acidic PR-2 (b-1,3-glucanase)
and PR-5 (thaumatin-like) were also detected in the leaves of
NCI-treated plants (Fig. 5A). By contrast, none of these transcripts could be detected in the leaves of water-treated control
plants (Fig. 5A). Treating NahG plants with NCI induced the
expression of PR-1, PR-2 and PR-5 genes as well (Fig. 5A).
This confirms that NCI does not require SA biosynthesis to
develop the disease resistance. These results indicate that NCI
is effective in inducing defense gene expression in SAindependent manner.
As foliar NCI-treatment showed variable efficacy depending on the pathogens tested, we examined PR gene expression
in this model as well. Foliar treatment with up to 5 mM NCI
failed to induce acidic PR-1 gene expression, while treatment
with 0.2 mM BTH was able to induce acidic PR-1 expression
(Fig. 5B). This result is consistent with the inability of foliar
treatment to induce disease resistance against Pst and O. lycopersici. To distinguish between direct NCI-stimulation of leaf
tissue and other signals released from root tissue in the activation of leaf tissue to express acidic PR-1, we analyzed acidic
PR-1 expression in leaf tissue infiltrated by NCI. Northern blot
analysis showed that acidic PR-1 gene expression was induced
in NCI-infiltrated tissue in a dose-dependent manner (Fig. 5C).
Leaf infiltration with even 0.5 mM NCI induced acidic PR-1
transcript accumulation in 3 d. By contrast, no transcripts were
detected in the water-infiltrated leaves of control plants. This
shows that NCI directly activates acidic PR-1 gene expression
in leaf tissue. This result is also observed in NahG plants,
which confirms that NCI-induced resistance is independent of
SA biosynthesis (Fig. 5C). Acidic PR-1 gene induction was
also observed in the more distant parts of the same leaf, in both
wild-type and NahG plants (Fig. 5C). Given these results and
the differential effects of foliar treatment on pathogens, NCI is
likely to be more easily incorporated by the root surface than
the leaf surface.
Discussion
In this paper, we demonstrate that NCI induces a broad
range of disease resistance in both tobacco and rice. In tobacco,
NCI enhances resistance against the viral pathogen TMV, the
bacterial pathogen Pst, and the fungal pathogen O. lycopersici,
but it dose not exhibit antibacterial activity. During resistance
induction, NCI itself induces PR gene expression in tobacco.
Fig. 6 Schematic pathways for induced resistance in tobacco and rice.
Therefore, NCI fulfills all the criteria of SAR inducer in
tobacco. Furthermore, measurement of SA accumulation and
use of NahG transgenic tobacco plants revealed that NCImediated resistance enhancement in tobacco does not require
SA; this suggests that NCI induces disease resistance by
triggering the signaling pathway at the same level as or downstream of SA, as do both BTH and INA.
Whether the same SAR signaling pathway functions in
both tobacco and rice has not yet been determined (Silverman
et al. 1995). Application of exogenous SA, the most plausible
signal compound for induced resistance in tobacco, is not
effective in rice. Application of JA has some protective effects
in rice, but suppresses plant growth (Schweizer et al. 1998).
Although the roles of SA, JA and other hormones in the development of rice SAR are unknown, three classes of chemicals
(which include INA, BTH and NCI) that activate disease resistance in rice are shown here to exert a similar effect in tobacco,
by stimulating downstream of SA. This supports the theory that
similar signaling pathways or at least several common components for induced resistance exist in tobacco and rice (Fig. 6).
This hypothesis is also supported by the fact that PBZ/BIT is
effective against rice blast disease.
In Arabidopsis, NPR1 is a key regulator of both SAR and
ISR, which functions at downstream of SA and JA, respectively (Cao et al. 1994, Delaney et al. 1995, Shah et al. 1997,
van Wees et al. 2000). The NPR1 homolog in rice is likely to
function in the development of resistance (Chern et al. 2001).
Since NCI exhibits all the necessary criteria for an SAR
inducer, as reported here, it would be interesting to examine
whether NCI requires NPR1 for SAR induction. Furthermore,
there are various mutants with defects in the SAR signaling
pathway in Arabidopsis. Therefore, a detailed mechanism of
NCI-induced resistance will likely be clarified using Arabidopsis, and this is currently under investigation.
It has been reported that NCI is much more effective
against rice blast disease than N-substituted 2-chloroisonicoti-
Chemically induced SAR in rice and tobacco
namide derivatives with other alkyl groups (Yoshida et al.
1990). In rice, NCI containing a 14C-labeled cyano group was
incorporated from the roots in plants 7 d after the treatment (up
to 4 ppm), thereby indicating that NCI was incorporated without degradation in soil (Yoshida et al. unpublished data). Thus,
the nucleophilic cyano group would be predicted to have some
effects during the interaction with the target protein, however,
exact effects have not been determined. The structures of NCI
and BIT contain similar moieties, such as an amide group
bound to an aromatic ring. NCI and INA have a common pyridine structure with chlorine atom and carbonyl group substitutions. Since NCI stimulates downstream of SA accumulation,
as shown here, investigating whether NCI and INA stimulate
the same target will elucidate a more detailed mechanism of
signal transduction in SAR development.
In the course of searching for chemicals effective against
rice pathogens, we have chosen the soil drench method because
of its practical applicability. This study demonstrates that NCI
is also effective in tobacco when applied by soil drenching.
Thus, NCI must be readily absorbed from the root surface in
rice, as is also the case in tobacco. Foliar treatment of NCI is
not effective in inducing PR gene expression and resistance
against Pst and O. lycopersici, but it is effective against TMV
that is inoculated with scratches on the leaf surface. Infiltration
of NCI into leaf tissue induces PR gene expression. Taken
together, we speculate that NCI is difficult to be incorporated
from the leaf surface. Possibly NCI activate SAR after being
carried to the cells that are sensitive to NCI, because its target
locates the lower point during the signaling pathway. However, we cannot exclude the possibility that some signals are
released from root tissue and mesophyll cells but not from epidermal cells, since it is not known how other SAR inducers
induce resistance systemically, whether by direct action after
diffusion to sensitive cells or by releasing some signal molecules. Differential effects of NCI treatment may be useful to
clarify this question in the future study. Using pure NCI, we
have identified a dose-dependent effect of NCI in rice, which
allowed for a comparison of efficacy with other chemicals. The
disease resistance induction activity of NCI was as potent as
that of BTH in rice, against both rice blast and bacterial blight
diseases. In tobacco, however, NCI exhibited more than 3-fold
less activity than BTH in cases of TMV and powdery mildew
infection. This probably reflects differences in these chemicals’ mode of action; these could be manifest in different
chemical absorption efficiencies from root surface, or in different structures or activities of the target proteins in these two
plants. NCI and other chemicals will likely be powerful tools
for investigating the SAR signaling pathway downstream of
SA as well as the broader similarities and differences between
SAR mechanisms in rice and tobacco.
829
Materials and Methods
Plant materials and treatment
O. sativa was grown in sterilized potting soil in pots (5´5´5 cm3)
inside a green house, at 25°C during the day and at 19°C during the
night, with 50–60% humidity. N. tabacum cv. Xanthi nc was grown in
sterilized potting soil (Kureha, Japan) in pots (6 cm diameter ´ 9 cm)
inside a growth chamber under a 16 : 8 h light : dark regimen, at 22°C,
with 60% humidity.
Rice pathogen infection assays
O. sativa cv. Aichiasahi was cultured in a pot (seven plants per
pot) in a greenhouse and 3-leaf stage plants were used for experiments. Pretreatment with various concentrations of NCI, BIT, BTH or
water was performed by soil drench application. Five d after pretreatment, challenge inoculation with M. grisea or X. oryzae pv. oryzae was
performed on pretreated leaves. In rice blast assay, plants were sprayed
with M. grisea conidia suspension (105 spores ml–1), kept under dark
condition with 100% humidity for 16 h, then incubated for 5 d in a
greenhouse (25°C). Infection on leaf-4 was calculated 5 d post inoculation. In rice bacterial blight assay, plants were cut at about 4 cm from
the tip of leaf-4, sprayed with cell suspension (109 colony-forming
units (CFU) ml–1) of X. oryzae pv. oryzae, and kept in a greenhouse
under the following conditions: 24°C and 70% humidity for 10 h without light; 29°C and 70% humidity for 14 h with light. The length of
bleached part of infected leaves was measured 12 d after challenge
inoculation.
Tobacco pathogen infection assays
Pretreatment of various concentrations of chemicals or water was
performed by soil drenching to 5-week-old plants. Foliar spraying was
performed to whole plants (5-week old) with 1 or 2.5 mM NCI,
0.2 mM BTH, or water.
In TMV infection assay, 5 d after pretreatment, a challenge inoculation with TMV was performed on the three leaves of pretreated
plants, before incubation at 22°C. Lesions were measured 5 d following the TMV inoculation.
A challenge inoculation with Pst was performed 5 d after pretreatment. Pst was cultured in nutrient broth containing rifampicin
(20 mg ml–1) at 28°C for 2 d, and a bacterial suspension was prepared
in 10 mM MgCl2 (2´105 CFU ml–1). Challenge inoculation was performed by infiltration of the bacterial suspension using a 1-ml syringe
without a needle. Leaf disks were taken from the infiltrated part of the
leaves at 0, 1, 2 and 4 dpi and three disks from a plant were combined
and homogenized in 10 mM MgCl2. The number of CFU was estimated by growth on nutrient broth agar plates after dilution. For each
time point, three plants were used, and two samples were prepared
from each plant.
O. lycopersici maintained on the tobacco leaves was transferred
onto new tobacco leaves for inoculation and was incubated at 20–
22°C, 60–70% humidity, in 16 : 8 h light : dark cycle. Sporulation was
seen after 1 week, and the white spores were powdered onto the pretreated plants placed together in a box (80´80´100 cm). Usually, about
25 cm2 of fully sporulated leaf was used to infect 12 pretreated plants.
The plants were incubated for 5 d as above and the degree of disease
symptoms was evaluated.
RNA analysis
Tobacco plants were treated with various concentrations of NCI,
0.2 mM BTH, or water by soil drench method, and the leaves were
harvested at various times after application. Total RNA was extracted
from frozen leaf samples of the plants using TRIzol reagent (Life
Technologies, Rockville, MD, U.S.A.) following the manufacturer’s
830
Chemically induced SAR in rice and tobacco
instructions. DNA fragment of the coding regions for tobacco PR
genes (Ward et al. 1991) were amplified by polymerase chain reaction
(PCR) from cDNA prepared from SA-treated tobacco. The PCR products were cloned into plasmid pCR2.1 (Invitrogen, Carlsbad, CA,
U.S.A.) and the nucleotide sequences were confirmed. 32P-labeled
cDNA probes were synthesized by random priming of these fragment
of acidic PR-1a, PR-2 and PR-5 genes. Total RNA samples were subjected to 1.2% agarose–1.1% formaldehyde gel electrophoresis and
transferred to a nylon membrane (Hybond N+, Amersham, Buckinghamshire, U.K.). After the transfer, RNA was cross-linked to the membrane using an UV linker (GS GENE LINKER, Bio-Rad, Hercules,
CA, U.S.A.). Hybridization and washing were performed as described
by Church and Gilbert (1984). Prehybridization and hybridization
were performed 68°C for 1 h or longer and 8 h or longer, respectively.
The membrane was washed twice with 2´ SSC containing 0.1% SDS
for 30 min at 68°C and then washed twice with 0.1´ SSC containing
0.1% SDS for 15 min at 68°C. The detection was performed with
BAS2500 image analyzer (Fujifilm).
Extraction and analysis of SA
Plants were treated by soil drenching with 5 mg pot–1 NCI or
water. Leaves were harvested from the treated plants 3 and 5 d after
treatment and SA and SAG levels were measured as previously
described (Yoshioka et al. 2001).
Acknowledgments
We would like to thank T. Hibi, Y. Takikawa, and J.A. Ryals, for
providing TMV, Pseudomonas strain, and NahG plant. We also thank
Y. Arimoto, T. Arie, and S. Gouthu for useful discussions. This work
was partially supported by a Grant-in-aid for scientific research on priority area (A) (No. 12052227) to H. N. from the Ministry of Education, Science and Culture of Japan.
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(Received January 21, 2002; Accepted May 9, 2002)