Finding new elicitors that induce resistance in rice to the white-backed planthopper Sogatella furcifera

Article abstract of DOI:10.1016/j.bmcl.2015.10.041

Herein we report a new way to identify chemical elicitors that induce resistance in rice to herbivores. Using this method, by quantifying the induction of chemicals for GUS activity in a specific screening system that we established previously, 5 candidate elicitors were selected from the 29 designed and synthesized phenoxyalkanoic acid derivatives. Bioassays confirmed that these candidate elicitors could induce plant defense and then repel feeding of white-backed planthopper Sogatella furcifera.

Full text of DOI:10.1016/j.bmcl.2015.10.041

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5601–5603
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Finding new elicitors that induce resistance in rice to the
white-backed planthopper Sogatella furcifera
a,
Xingrui He a,y, Zhaonan Yu b,y, Shaojie Jiang a, Peizhi Zhang c, Zhicai Shang a, Yonggen Lou b, , Jun Wu
⇑
⇑
a Department of Chemistry, Zhejiang University, Hangzhou 310027, China
b National Key Laboratory of Rice Biology, Institute of Insect Science, Zhejiang University, Hangzhou 310058, China
c School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou 310023, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we report a new way to identify chemical elicitors that induce resistance in rice to herbivores.
Using this method, by quantifying the induction of chemicals for GUS activity in a specific screening sys-
tem that we established previously, 5 candidate elicitors were selected from the 29 designed and synthe-
sized phenoxyalkanoic acid derivatives. Bioassays confirmed that these candidate elicitors could induce
plant defense and then repel feeding of white-backed planthopper Sogatella furcifera.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 20 May 2015
Revised 12 October 2015
Accepted 15 October 2015
Available online 23 October 2015
Keywords:
Chemical elicitors
White-backed planthopper
GUS activity
Structure–activity relationships
Chemical insecticides have a pivotal role in crop protection.
However, the extensive application of these insecticides not only
causes severe environmental and farm produce pollution but also
damages the ecosystem and thus results in, for example, the resur-
gence of herbivores and the reduction in populations of the natural
enemies of herbivores and biodiversity. Therefore, developing safe
and effective methods to control insect pests is essential.1,2
To protect themselves from damage by invaders, plants in nat-
ure have evolved a series of defense mechanisms, including
induced defenses that are activated by herbivore attack.3–6 Studies
on the mechanisms underlying herbivore-induced plant defense
have revealed that many compounds with low molecular weight,
such as herbivore-associated molecule patterns, jasmonic acid
(JA), salicylic acid (SA) and ethylene, play an important role in this
process. Exogenous application of these compounds or their syn-
thetic analogues, defined as chemical elicitors, can induce plant
defense responses and thus increase the resistance of plants to her-
bivores.7,8 Since the chemical elicitors themselves are not toxic to
herbivores or their natural enemies and decompose easily in nat-
ure, the development of safe plant protection agents based on
chemical elicitors is highly desired.
elicitors.9,10 With the methods that chemical genetics makes avail-
able, we have established a specific high-throughput screening sys-
tem, which contains a 1.55-kb promoter region of the S-linalool
synthase gene (LISp), fused to a b-glucuronidase (GUS) reporter
gene.7 With this system, the potential chemical elicitors can be iden-
tified according to the GUS activity that they induce.7 Thus far, we
have identified an herbicide, 2,4-D (2,4-dichlorophenoxyacetic acid),
that is a chemical elicitor and found that it has potential to help
control the rice brown planthopper Nilaparvata lugens in the field.7
In order to develop new chemical elicitors that are safe protec-
tion agents for controlling insect pests, we designed and synthe-
sized 29 phenoxyalkanoic acid derivatives and explored the
relationship between their structures and the levels of GUS activity
that they induced. Moreover, we investigated the effect of these
potential elicitors on transcript levels of two defense-related genes
in rice, a mitogen-activated protease kinase (MPK) gene, OsWPK3,
and a linalool synthase gene, OsLIS, whose encoding enzyme cat-
alyzes the production of a volatile monoterpenoid linalool; both
of these represent early and late events, respectively, in the herbi-
vore-induced plant defense responses.11,12 Finally, we measured
the effect of treatment with the potential elicitors on the resistance
of rice to the white-backed planthopper Sogatella furcifera, one of
the most important rice insect pests in Asia.13,14 Here, we report
our findings.
Chemical genetics has been used widely to develop drugs and
offers
a powerful toolkit with which to discover chemical
In this study, we used the high-throughput screening system9,10
to evaluate the induction activities of 29 phenoxyalkanoic acid
derivatives. GUS activity was quantified in the roots of transgenic
⇑
Corresponding authors. Tel./fax: +86 571 88982622 (Y.L.); tel.: +86 571
87951352; fax: +86 571 87951895 (J.W.).
E-mail addresses: yglou@zju.edu.cn (Y. Lou), wujunwu@zju.edu.cn (J. Wu).
These authors contributed equally to this work.
y
http://dx.doi.org/10.1016/j.bmcl.2015.10.041
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

5602
X. He et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5601–5603
Table 1
rice plants that had been treated with one of the tested compounds
at a concentration of 5 mg Lꢀ1, using the same method as described
in Xin et al.7 The relative induction of GUS activity in the roots of
plants treated with each compound compared to in the roots of
the control (non-treated) plant is summarized in Tables 1 and 2.
Among the tested chemicals derived from phenoxyacetic acid
and 24 ring-substituted phenoxyacetic acid (Table 1), five
compounds elicited GUS levels that were higher than those were
found in the control. Moreover, levels of GUS activity in four mono-
substituted phenoxyacetic acids—4-methylphenoxyacetic acid,
4-iodophenoxyacetic acid, 4-bromophenoxyacetic acid and 4-fluo-
rophenoxyacid acid—were higher than the levels found in phenoxy-
acetic acid by 1.63-, 1.34-, 1.34- and 1.21-fold, respectively. Within
the family of these monosubstituted phenoxyacetic acids, 2- or 3-
position substitutions did not increase the level of GUS activity com-
pared with level of phenoxyacetic acid (e.g., 2-methylphenoxyacetic
acid < phenoxyacetic acid; 3-methylphenoxyacetic acid < phenoxy-
acetic acid). With the exception of 4-ethylphenoxyacetic acid,
4-tert-butylphenoxyacetic acid, 4-methoxyphenoxyacetic acid,
4-nitrophenoxyacetic acid and 4-acetamidophenoxyacetic acid, the
4-substituted halogphenoxyacetic acids and 4-methylphenoxy-
acetic acid induced higher levels of GUS activity than did phenoxy-
acetic acid. Substitution with strong electron-withdrawing
characteristics, such as nitro group at 4-position, and di-substitu-
tion, reduced activity (e.g., 4-chloro-2-methylphenoxyacetic
acid < phenoxyacetic acid < 4-methylphenoxyacetic acid).
Relative induction of GUS activity in rice by phenoxyacetic acid and ring-substituted
phenoxyacetic acids
Compound
Substituent Relative
induction
of GUS
activitya
Control
2-Phenoxyacetic acid (1a)
1.00
1.43⁄⁄
Position 2 substitution
2-Methylphenoxyacetic acid (1b)
2-Isopropylphenoxyacetic acid (1c)
2-Methoxyphenoxyacetic acid (1d)
2-Fluorophenoxyacetic acid (1e)
2-Chlorophenoxyacetic acid (1f)
2-Bromophenoxyacetic acid (1g)
2-Iodophenoxyacetic acid (1h)
2-Nitrophenoxyacetic acid (1i)
2-Acetamidophenoxyacetic acid (1j)
2-Acetylphenoxyacetic acid (1k)
CH3
iso-Propyl
CH3O
F
Cl
Br
I
NO2
CH3CONH
CH3CO
0.99
1.18
1.23
0.87
0.93
0.67⁄
1.02
1.11
1.09
1.05
Position 3 substitution
3-Methylphenoxyacetic acid (1l)
3-Methoxyphenoxyacetic acid (1m)
3-Chlorophenoxyacetic acid (1n)
3-Bromophenoxyacetic acid (1o)
CH3
CH3O
Cl
0.69⁄
1.06
0.88
1.17
Br
Position 4 substitution
4-Methylphenoxyacetic acid (1p)
4-Ethylphenoxyacetic acid (1q)
4-tert-Butylphenoxyacetic acid (1r)
4-Methoxyphenoxyacetic acid (1s)
4-Fluorophenoxyacetic acid (1t)
4-Bromophenoxyacetic acid (1u)
4-Iodophenoxyacetic acid (1v)
4-Nitrophenoxyacetic acid (1w)
4-Acetamidophenoxyacetic acid (1x)
CH3
CH3CH2
tert-Butyl
2.33⁄⁄
0.62⁄⁄
1.19
CH3O
F
Br
I
0.95⁄⁄
1.91⁄⁄
1.92⁄⁄
0.94
The GUS activity levels induced by the substituted phenoxybu-
tanoic acids, 1-naphthyloxypropanoic acid and phenoxyacetamide
are listed in Table 2. After the length of the side chain between
phenoxy moiety and carboxylic acid was increased, the levels of
GUS activity diminished dramatically. This result suggests that an
increase in the chain length does not improve the level of activity.
In addition, phenoxyacetamide elicited lower levels of GUS activity
than phenoxyacetic acid did, indicating that the carboxylic acid
group is necessary for high activity.
1.72
NO2
CH3CONH
1.00
Mixed halogen and alkyl substitutions
4-Chloro-2-methylphenoxyacetic acid (1y)
2-CH3-4-Cl
1.16
a
GUS activity level induced by the tested compound (5 mg Lꢀ1) divided by the
level induced by the control 48 h after treatment. Asterisks indicate significant
differences between treatments and controls (⁄P <0.05, ⁄⁄P <0.01, Student’s t-tests).
From the GUS activity levels of the above phenoxyacetic acid
derivatives, we can deduce that levels of GUS activity are increased
when specific conditions are met: (1) Enhanced activity is limited
to substitution at 4-position; Substitutions at 2- or 3-position
decreased induction activity. (2) Substitution is sensitive to the
induction of GUS activity, and the small methyl group, a fluorine
atom, a bromine atom and an iodine atom enhance activity. (3)
The length of the chain between phenoxy moiety and carboxylic
acid is one of the important factors influencing the induction
potency for GUS activity, and the methylene bridge enhances activ-
ity. (4) A carboxylic acid group is necessary and can increase activ-
ity dramatically.
Table 2
Relative induction of GUS activity in rice by ring-substituted phenoxybutanoic acids,
naphthyloxypropanoic acid, and phenoxyacetamide
Compound
Relative induction of GUS
activity*
Control
1.00
0.92
4-(2,4-Dichlorophenoxy)butanoic acid
(2a)
4-(2,6-Diisopropylphenoxy)butanoic acid
(2b)
0.98
2-(1-Naphthyloxy)propanoic acid (2c)
Phenoxyacetamide (2d)
0.94
1.06
To confirm whether screening out the phenoxyacetic acid
derivatives can elicit defense responses, we investigated the
expression levels of the two defense-related genes, OsLIS and
OsMPK3, in the stems of rice plants whose roots were treated with
one of the following compounds 1a, 1p, 1t, and 1u at a concentra-
tion of 5 mg Lꢀ1. The result showed that all of the four compounds
elicited increases in transcript levels of the two genes: levels of
OsMPK3 transcripts increased at 12 and 24 h after the start of treat-
ment, whereas levels of OsLIS transcripts mainly increased 24 h
after treatment (Fig. 1). This suggests that the screened chemical
elicitors can profoundly induce defense responses in rice.
To further explore the effect of these chemical elicitor-induced
defense responses on herbivore performance, we investigated the
feeding preference of adult S. furcifera females in the lab for plants
treated with one of these chemical elicitors and control plants. The
results showed that S. furcifera female adults were more frequently
found on control plants than on plants whose roots had been trea-
ted with one of the four chemical elicitors 1a, 1p, 1t, and 1u at a
GUS activity level induced by the tested compound (5 mg Lꢀ1) divided by the
level induced by the control 48 h after treatment.
*
concentration of 5 mg Lꢀ1 (Fig. 2), suggesting the treated plants
were repellent to the herbivore.
Moreover, the tested chemical elicitors themselves were not
repellent to the herbivore: the number of female adults that stayed
on cotton balls containing 500
l
l of 30% sucrose solution was equal
to those that stayed on cotton balls containing 500
l
l of 5 mg Lꢀ1
the tested chemical elicitor in the solution (Fig. 3).Taken together,
the data demonstrate that the repellent role of the elicitor-treated
plants to the herbivore comes from the defense responses induced
by the elicitors. Among the tested four chemical elicitors, 1p
induced the highest and longest-lasting level of repellency in the
herbivore: it reached about 50% at 48 h after treatment (Fig. 2).

X. He et al. / Bioorg. Med. Chem. Lett. 25 (2015) 5601–5603
5603
0.6
0.4
0.2
12
C
12
9
c
C
1a
1p
1t
**
1a
1p
**
*
9
6
1u
*
6
*
3
3
1
2
4
4
8
8
24 48
1
1
2
4
4
8
8
24 48
12
12
C
1t
C
1u
0.0
0.4
9
6
3
9
6
3
12
24
c
1a
1p
1t
0.3
0.2
0.1
0.0
1u
1
2
24 48
2
24 48
Time᧤K ꢀ
**
*
**
*
**
**
Figure 3. Mean number of female Sogatella furcifera adults (+SE, n = 10) on pairs of
cotton balls, a cotton ball with 1 ml of one of the four compounds, 2-phenoxyacetic
acid (1a), 4-methylphenoxyacetic acid (1p), 4-fluorophenoxyacetic acid (1t), and 4-
bromophenoxyacetic acid (1u), at a concentration of 5 mg Lꢀ1 versus a cotton ball
with 1 ml of the buffer (C), 1–48 h after exposure.
**
**
12
24
Time (h)
demonstrate that the ability of a chemical elicitor to induce GUS
activity is correlated with its ability to induce resistance in rice
to S. furcifera, suggesting the value of using the relative induction
of GUS activity as a screening tool for identifying new chemical
elicitors and developing insect control agents. The chemical
elicitors screened out here could be exploited as induced insect
repellants against S. furcifera. Future research should explore the
effectiveness of these compounds for controlling S. furcifera in
the field.
Figure 1. Mean expression levels (+SE, n = 5) of OsLis (a) and OsWPK3 (b) in stems
of plants whose roots were treated with one of the four compounds, 2-
phenoxyacetic acid (1a), 4-methylphenoxyacetic acid (1p), 4-fluorophenoxyacetic
acid (1t), and 4-bromophenoxyacetic acid (1u) at a concentration of 5 mg Lꢀ1. (C)
Non-manipulated. Asterisks indicate significant differences between treatments
and controls at each time point (⁄P <0.05, ⁄⁄P <0.01, Student’s t-tests).
12
9
12
9
C
C
1a
1p
*
**
**
**
**
**
*
*
Acknowledgements
**
The study was jointly sponsored by the Special Fund for
Agro-scientific Research in the Public Interest (201403030), the
National Natural Science Foundation of China (No. 31471807),
and the China Agriculture Research System (CARS-01-21).
6
6
3
3
1
2
4
8
24 48
1
1
2
4
8
24 48
12
12
C
1t
**
C
Supplementary data
1u
*
*
**
**
**
**
9
6
3
9
6
3
*
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.10.
041.
*
References and notes
1
2
4
8
24 48
2
4
8
24 48
Time᧤K ꢀ
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a
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In conclusion, among the compounds tested, only monosubsti-
tuted phenoxyacetic acids with a methyl group, a fluorine atom, a
bromine atom or an iodine atom in the 4-ring position are more
active in inducing GUS activity than phenoxyacetic acid is. We
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