The Plant Journal The rice hydroperoxide lyase OsHPL3 ...sippe.ac.cn/zuhuahe/publication/Tong...

The rice hydroperoxide lyase OsHPL3 functions in defenseresponses by modulating the oxylipin pathway

Xiaohong Tong1,2,†, Jinfeng Qi1,†, Xudong Zhu3,†, Bizeng Mao1, Longjun Zeng2, Baohui Wang1, Qun Li2, Guoxin Zhou1,

Xiaojing Xu4, Yonggen Lou1,* and Zuhua He1,2,*1College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China,2National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology

and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China,3China National Rice Research Institute, 359 Tiyuchang Road, Hangzhou 31006, China, and4National Engineering Center for BioChip at Shanghai, Shanghai 201203, China

Received 9 January 2012; revised 15 April 2012; accepted 17 April 2012; Published online 18 June 2012.

*For correspondence (e-mails [email protected] or [email protected]).†These authors contributed equally to this work.

SUMMARY

As important signal molecules, jasmonates (JAs) and green leaf volatiles (GLVs) play diverse roles in plant

defense responses against insect pests and pathogens. However, how plants employ their specific defense

responses by modulating the levels of JA and GLVs remains unclear. Here, we describe identification of a role

for the rice HPL3 gene, which encodes a hydroperoxide lyase (HPL), OsHPL3/CYP74B2, in mediating plant-

specific defense responses. The loss-of-function mutant hpl3-1 produced disease-resembling lesions spreading

through the whole leaves. A biochemical assay revealed that OsHPL3 possesses intrinsic HPL activity,

hydrolyzing hydroperoxylinolenic acid to produce GLVs. The hpl3-1 plants exhibited enhanced induction of JA,

trypsin proteinase inhibitors and other volatiles, but decreased levels of GLVs including (Z)-3-hexen-1-ol.

OsHPL3 positively modulates resistance to the rice brown planthopper [BPH, Nilaparvata lugens (Stal)] but

negatively modulates resistance to the rice striped stem borer [SSB, Chilo suppressalis (Walker)]. Moreover,

hpl3-1 plants were more attractive to a BPH egg parasitoid, Anagrus nilaparvatae, than the wild-type, most

likely as a result of increased release of BPH-induced volatiles. Interestingly, hpl3-1 plants also showed

increased resistance to bacterial blight (Xanthomonas oryzae pv. oryzae). Collectively, these results indicate

that OsHPL3, by affecting the levels of JA, GLVs and other volatiles, modulates rice-specific defense responses

against different invaders.

Keywords: Oryza sativa L., oxylipin pathway, jasmonates, green leaf volatiles, hydroperoxide lyase, herbivore-

induced plant defense.

INTRODUCTION

Natural co-evolution has armed plants with sophisticated

defense machinery against pathogens and insect pests,

including not only pre-existing defense barriers but also

inducible defense responses such as accumulation of

defense compounds induced by herbivore and pathogen

attack. The activation of inducible defenses depends on a

complicated signaling network, in which the oxylipin path-

way plays a central role (Bostock, 2005; Browse and Howe,

2008; Howe and Jander, 2008). Oxylipins including jasmo-

nates (JAs) and other related chemicals are synthesized via

the early precursor linolenic acid that is oxygenated by

13-lipoxygenase (13-LOX) into hydroperoxy polyunsatu-

rated fatty acid, which is the common substrate for several

enzymes of the oxylipin pathway, such as allene oxide syn-

thase (AOS) and hydroperoxide lyase (HPL) (Feussner and

Wasternack, 2002). The products of the AOS and HPL cas-

cades are JAs and green leaf volatiles (GLVs), respectively,

both of which are vital signaling compounds that play dis-

tinct roles in plant direct and indirect defenses (Kessler and

Baldwin, 2001; Shiojiri et al., 2006; Browse and Howe, 2008;

Allmann and Baldwin, 2010).

As a monocot plant, rice (Oryza sativa L.) has been

adopted as a model crop for studying defense responses

against pathogens and insects. However, it was only

recently recognized that the JA signaling pathway plays

important roles in defense against pest insects in rice.

ª 2012 The Authors 763The Plant Journal ª 2012 Blackwell Publishing Ltd

The Plant Journal (2012) 71, 763–775 doi: 10.1111/j.1365-313X.2012.05027.x

Knockdown of OsHI-LOX expression by an antisense trans-

gene (as-lox) reduced JA and trypsin proteinase inhibitors

(TrypPIs) but not GLV levels induced by the rice striped stem

borer (SSB), Chilo suppressalis (Walker), and the rice brown

planthopper (BPH), Nilaparvata lugens (Stal), resulting in

improved larval performance of SSB and the rice leaf folder

Cnaphalocrocis medinalis on as-lox plants (Zhou et al.,

2009). In contrast, as-lox plants showed increased resistance

to BPH, a phloem-feeding herbivore, and accumulated

higher levels of BPH-induced H2O2 and salicylic acid (SA),

leading to increased hypersensitive response-like cell death,

indicating that OsHI-LOX is involved in herbivore-induced

JA biosynthesis and plays distinct roles in rice resistance to

chewing and phloem-feeding herbivores (Zhou et al., 2009).

Recently, we also dissected the first step of the oxylipin

pathway, the release of linolenic acid from chloroplast

membranes by chloroplast-localized Phospholipases D

Os-PLDa4 and OsPLDa5 in rice. Antisense-mediated sup-

pression of OsPLDa4/5 expression in as-pld plants resulted

in reduced induction of linolenic acid, JA, GLVs and ethyl-

ene. The impaired oxylipin and ethylene signaling in as-pld

plants decreased the levels of herbivore-induced TrypPIs

and other volatiles, leading to improved performance of SSB

and BPH, and reduced attractiveness to a larval parasitoid of

SSB, Apanteles chilonis (Qi et al., 2011). These studies

indicate that JA plays positive roles in rice resistance to

the chewing herbivore SSB, but a negative role in resistance

to the phloem feeder BPH, while GLVs positively mediates

resistance to both the herbivores in rice.

Green leaf volatiles are formed through the HPL-medi-

ated cascade of oxylipin metabolism during tissue disrup-

tion upon biotic or abiotic stress. It has been postulated

that GLVs are important signals that are perceived by

neighboring plants and other organisms including herbi-

vores and their natural enemies (Matsui, 2006). When over-

expressed in transgenic Arabidopsis, the HPL gene from

bell pepper (Capsicum annuum L.) enhanced GLV

production, resulting in increased attractiveness to the

parasitic wasp Cotesia glomerata, whereas HPL antisense

transgenic Arabidopsis produced fewer GLVs and showed

reduced attractiveness to parasitoids, resulting in de-

creased indirect herbivore resistance (Shiojiri et al., 2006).

Moreover, HPLs also affect the levels of JAs and other

metabolites of the AOS cascade via common substrates.

For instance, in Nicotiana attenuata, antisense inhibition of

NaHPL decreased the levels of GLVs but increased JA

levels (Halitschke et al., 2004). Therefore, HPLs may mod-

ulate specific defense responses to various invaders by

affecting the levels of JAs and GLVs in diverse plant

species. Moreover, production of TrypPIs is modulated by

both JA and GLVs, although other herbivore-induced

volatiles are only affected by the JA pathway in N. atten-

uata (Halitschke et al., 2004). However, the activity of

TrypPIs in rice is mainly induced by JA but not GLVs (Qi

et al., 2011), suggesting complexity and divergence of the

oxylipin pathway among diverse plant species.

It is well known that the SA pathway antagonizes the JA

pathway in defense responses in diverse plant species.

Generally, the JA pathway is considered to function mainly

against a broad range of phloem feeders and chewing

herbivores (Ellis et al., 2002; Zarate et al., 2007; Howe and

Jander, 2008; Wang et al., 2008), whereas the SA pathway

functions in defense response against pathogens (Wilder-

muth et al., 2001; Lu, 2009). Interestingly, some phloem-

feeding insects such as the silver leaf whitefly Bemisia tabaci

and aphids also activate the SA pathway by a decoy strategy,

suppressing the JA-mediated defense in some plant species

(Walling, 2000, 2008; Thompson and Goggin, 2006; Zarate

et al., 2007). Several studies also showed that SA-mediated

defenses act against phloem feeders (Mewis et al., 2005;

Pegadaraju et al., 2005; Thompson and Goggin, 2006).

Therefore, the roles of JA and SA in insect resistance

probably depend on plant species (Goggin, 2007). Our

previous study showed that the SA pathway suppressed

the JA-mediated defense against herbivores in rice (Yuan

et al., 2007). Recently, Gomi et al. (2010) found that infesta-

tion by the rice white-backed planthopper Sogatella furcifera

induced expression of OsHPL2 to elicit production of vola-

tiles that enhance rice resistance to bacterial blight, which

usually depends on the SA pathway (Yuan et al., 2007).

However, genetic evidence for cross-talk between the SA

and JA pathways against pathogens and herbivores is still

limited in rice.

The rice genome contains three HPLs that metabolize

different hydroperoxides (Chehab et al., 2006). However,

whether HPLs play a role in rice defense remains unknown.

In this study, we report that the rice HPL3 gene is critical for

herbivore-induced plant defense responses. OsHPL3 (Che-

hab et al., 2006) positively modulates the production of

GLVs but negatively affects other volatiles and TrypPI

activity as well as JA biosynthesis through substrate

competition. We show that OsHPL3 acts as a negative

regulator of plant resistance to the chewing herbivore SSB,

but a positive regulator of defense against the phloem

feeder BPH. OsHPL3 also functions in indirect resistance to

BPH through attractiveness to Anagrus nilaparvatae, an egg

parasitoid of BPH. We propose that OsHPL3 modulates rice

direct and indirect defense responses by affecting the levels

of JA and GLVs.

RESULTS

Characterization of the hpl3-1 mutant and map-based

cloning of OsHPL3

We have studied hormone-mediated defense against insects

and pathogens in rice through forward and reverse genetic

approaches (Yuan et al., 2007; Yang et al., 2008; Zhou et al.,

2009; Qi et al., 2011). The rice hpl3-1 mutant, which was

764 Xiaohong Tong et al.

ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 763–775

isolated from the mutation pool of the japonica variety

Zhonghua 11 (Zhu et al., 2003), displayed necrotic lesions in

the leaves of 2-week-old seedlings. These necrotic lesions

gradually extended during plant growth, resulting in large

brown lesions on mature leaves (Figure 1a,b). Compared

with the wild-type (WT) plants, the hpl3-1 plants developed

fewer tillers and decreased plant height, panicle size and

grain weight (Figure S1). The development of lesions on

hpl3-1 was light-dependent (Figure S1), suggesting that the

cell death signal may be induced by light.

To identify the mutant gene, we performed map-based

cloning using the F2 population from a cross of the mutant

with an indica variety. The candidate gene was delimited to

a genomic region of chromosome 2 between markers T4

and T6 on a single bacterial artificial chromosome (BAC

AP004752) (Figure 1c). This region contains six putative

genes. Sequencing the candidates revealed that Os02g0

110200, which encodes an HPL (OsHPL3, accession number

AY340220; Chehab et al., 2006) with 487 amino acids, was

disrupted by insertion of a transposon-like element of

approximately 700 bp between nucleotides 518 and 519

(Figure 1c,d). The insertion probably results in loss of

Os02g0110200 functions, as its transcripts and encoded

products were undetectable in hpl3-1 (Figure 1e,f). Further-

more, a complementation construct containing the WT

OsHPL3 genomic region with its own promoter comple-

mented the hpl3-1 phenotypes (Figure 1a,b), confirming

that the mutation in OsHPL3 is responsible for the mutant

phenotypes. Quantitative PCR analysis showed that OsH-

PL3 is highly expressed in the seedlings, leaves and

panicles (Figure 2a), and its expression was induced by

wounding, BPH and SSB infestation but not by JA and SA

(Figure 2b–f).

The rice genome encodes three HPLs, OsHPL1, OsHPL2

and OsHPL3, which belong to the CYP74C (OsHPL1 and

OsHPL2) or CYP74B (OsHPL3) sub-families. As shown in

Figure S2, OsHPL3 shares limited homology with other

HPLs, including OsHPL1, OsHPL2 and an Arabidopsis HPL

that modulates biosynthesis of GLVs and JA in Arabidopsis

and tobacco (Halitschke et al., 2004; Shiojiri et al., 2006).

(a)

(c)

(d) (e) (f)

(b)Figure 1. Phenotypes of the hpl3-1 mutant and

map-based cloning of the OsHPL3 gene.

(a) Adult WT plants, hpl3-1 plants, genetically

complemented plants (hpl3-1 + 1300-OsHPL3)

and plants transformed with the empty vector

control (hpl3-1 + 1300).

(b) Lesion-mimic phenotypes of complementa-

tion plants. Four representative independent

complementation lines (hpl3-1 + 1300-OsHPL3)

are shown.

(c) Map-based cloning of the OsHPL3 gene. The

markers for PCR-based mapping are listed in

Table S1.

(d) PCR detection of the hpl3-1 locus. Note that a

transposable element was inserted into the

OsHPL3 gene in hpl3-1 as indicated in (c).

(e) Expression of OsHPL3 in WT and hpl3-1 plants

by RT-PCR analysis. Rice Ubi-1 (OsUBI) was used

as an internal control.

(f) Western blot analysis was performed with

anti-OsHPL3 antibodies to detect protein levels

of OsHPL3.

Hydroperoxide lyase functions in rice defense 765


Mutation in OsHPL3 reduces wound-induced GLV emission

but increases JA accumulation

As HPLs function in the oxylipin metabolism pathway, it is

possible that OsHPL3 may compete with AOS for the same

substrate, hydroperoxylinolenic acid, to generate GLVs,

whereas AOS generates JA (Figure 3a). It has been shown

previously that OsHPL1 and OsHPL2 metabolize 9-/13-

hydroperoxides, whereas OsHPL3 exclusively metabolizes

13-hydroperoxylinolenic acid (13-HPOT) generated by

13-LOX (Chehab et al., 2006). To examine the substrate

preferences of OsHPL3, we performed enzyme assays with

yeast microsomes harboring the protein in the presence of

either 9-/13-HPOT or 9-/13-hydroperoxylinoleic acid (9-/13-

HPOD) as substrates. These data, in agreement with those

previously published (Chehab et al., 2006), clearly show

that OsHPL3 is exclusively active on 13-HPOT (Figures 3b

and S3). Furthermore, we also assayed HPL activity in leaf

extracts as described by Duan et al. (2005), and found that

extracts from WT plants had stronger enzymatic activity on

13-HPOT but not 13-HPOD, 9-HPOT and 9-HPOD than

extracts from hpl3-1 plants (Figure 3c), strongly suggesting

that OsHPL3 is a major HPL that exclusively metabolizes

13-HPOT in rice leaves.

To further investigate the OsHPL3 function in oxylipin

metabolism, we profiled compounds of the oxylipin path-

way in rice. Consistent with the HPL activity of OsHPL3,

wound-induced levels of the GLVs (Z)-hexenal and (Z)-3-

hexen-1-ol were significantly decreased in hpl3-1 plants

compared with those in WT plants (Figure 4a,b). In

Figure 2. Tissue-specific expression and induction of the OsHPL3 gene.

Transcript levels of OsHPL3 were detected by real-time PCR in WT plants subjected to wounding, SSB and BPH feeding, and JA and SA treatments.

(a) Expression levels of OsHPL3 in various tissues of plants. Note that OsHPL3 has high expression levels in seedlings.

(b–f) Induction of OsHPL3 in stems of plants treated by wounding (b), infestation with BPH (c) and SSB (d), JA (e) and SA (f), showing that OsHPL3 is highly inducible

by wounding, SSB and BPH infestation.

Rice ACTIN1 was used as an internal control. Values are means � SE and were obtained in one experiment with three biological replicates, and similar results were

obtained in two independent experiments. Asterisks indicate significant differences between treatments and respective controls at each time point (*P < 0.05,

**P < 0.01; Student’s t test).



contrast, both basal and SSB-induced JA levels were

obviously higher in hpl3-1 plants than those in WT plants

(Figure 4c), but little difference was observed for BPH-

induced JA levels between WT and hpl3-1 plants (Fig-

ure 4d). Consistent with the elevated JA levels, expression

levels of AOS2 (AY062258) and JAmyb (AY026332) were

significantly higher in hpl3-1 plants than in WT plants

(Figure S4). These results together indicate that OsHPL3,

as an HPL, not only controls GLV production but also

affects JA biosynthesis, because HPLs use a common

(a)

(b)

Figure 3. HPL activity of OsHPL3 towards different substrates in vitro.

(a) The major oxylipin pathway. Hydroperoxylinoleic acid is catabolized by HPL3 to form (Z)-3-hexenal or by AOS to form JA and its derivatives. (Z)-3-hexenal is

further converted to (Z)-3-hexen-ol by ADH.

(b) In vitro enzyme activity assays of recombinant OsHPL3 using 9-/13-hydroperoxides with the direct reaction (Chehab et al., 2006). The activity of OsHPL3 with the

yeast microsomal protein (left), using a yeast microsomal preparation containing the rice EUI P450 that has no HPL activity as a control (right), was measured by

monitoring the loss of absorbance of substrate at 234 nm.

(c) OsHPL3 activity in leaf extracts of WT and hpl3-1 plants using 9-/13- hydroperoxides with the indirect reaction (Duan et al., 2005). The activity of leaf extracts of WT

plants (left) and leaf extracts of hpl3-1 plants (right) was measured by monitoring the loss of absorbance of substrate at 340 nm (coupled with ADH), using

background oxidation of NADH without the substrates as a control.

Note that OsHPL3 exhibited HPL activity with 13-HPOT, but not 13-HPOD, 9-HPOD or 9-HPOT. LOX, lipoxygenase; HPL, hydroperoxide lyase; AOS, allene oxide

synthase; ADH, alcohol dehydrogenase; 13-HPOT, 13-hydroperoxylinolenic acid; 13-HPOD, 13-hydroperoxylinoleic acid; 9-HPOT, 9-hydroperoxylinolenic acid; 9-

HPOD, 9-hydroperoxylinoleic acid. The results shown were obtained in one experiment with three biological replicates, and similar results were obtained in three

independent experiments.



substrate with the AOS cascade that produces JA (Hali-

tschke et al., 2004).

OsHPL3 affects induction of TrypPIs and other volatiles

To test the effect of OsHPL3 on rice defense, we measured

the activity of TrypPIs, which are important defense-related

proteins in rice against chewing herbivores such as SSB

(Zhou et al., 2009). No significant difference was observed in

the basal levels of TrypPIs between WT and hpl3-1 plants,

whereas, when the plants were infested by SSB for 1, 3 or

5 days, the levels of TrypPIs in hpl3-1 were significantly

higher than those in WT plants (Figure 5a). Moreover, as

other plant volatiles also play roles in rice defense, including

attracting natural enemies of BPH (Lou et al., 2005), we also

collected and analyzed other volatiles emitted from hpl3-1

and WT plants with or without infestation by BPH. Although

there was little difference in basal volatile release between

WT and hpl3-1 plants, BPH-infested hpl3-1 plants emitted

much higher amounts of these volatiles than WT plants

did (Figures 5b and S5). Emission of eight compounds [2-

heptanol, methyl benzoate, linalool, (E)-a-bergamotene,

sesquisabinene A, a-curcumene, b-bisabolene and (E)-c-bi-

sabolene] was significantly increased in hpl3-1 plants, but

the amount of one chemical, a-pinene, was reduced in hpl3-1

plants compared with WT plants (Figures 5b and S5). These

results indicate that OsHPL3 also affects the induction of

TrypPIs and other volatiles.

OsHPL3 modulates rice resistance to various herbivores

Our previous studies have shown that JA and GLVs play

important roles in rice defenses against herbivores (Zhou

et al., 2009; Lu et al., 2011; Qi et al., 2011). To determine

whether OsHPL3 mediates rice resistance to herbivores, we

first assessed SSB performance on hpl3-1 and WT plants. As

shown in Figure 5(c), SSB caterpillars gained more mass on

WT plants than on hpl3-1 plants. By day 13, the mean mass

of caterpillars that fed on WT plants was 1.93-fold higher

than those on hpl3-1 plants (a statistically significant differ-

ence). Moreover, SSB larva suffered significantly higher

mortality on hpl3-1 plants than WT plants (Figure 5c, inset),

indicating that OsHPL3 negatively modulates rice resistance

to SSB.

We then determined the influence of OsHPL3 on the host

preference and performance of BPH, a phloem-feeding

herbivore of rice. We found that both BPH nymphs and

female adults preferred to feed on hpl3-1 plants over WT

plants (Figure 6b,e), and female adults laid significantly

more eggs on the former than the latter (Figure 6e, inset). In

addition, the developmental duration of BPH eggs was

significantly shorter on hpl3-1 plants than on WT plants

(Figure 6a). The amount of honeydew excreted per day by a

BPH female adult feeding on hpl3-1 plants was significantly

higher (1.69-fold) than that on WT plants (Figure 6a, inset). In

contrast, BPH nymphs (Figure 6c,d) and female adults

Figure 4. Levels of (Z)-3-hexenal, (Z)-3-hexen-1-

ol, JA and SA in hpl3-1 and WT plants following

different treatments.

(a,b) Levels of (Z)-3-hexenal (a) and (Z)-3-hexen-

1-ol (b) (peak area/mg fresh mass) in leaves of

hpl3-1 and WT plants before (control) and after

the leaves were cut into small pieces (wound).

(c,d) Levels of JA in stems of hpl3-1 and WT

plants at 0, 1.5 and 3 h after SSB infestation (c),

and levels of JA in leaf sheaths of hpl3-1 and WT

plants at 0, 8 and 48 h after BPH infestation (d).

(e,f) Levels of SA in stems of hpl3-1 and WT

plants at 0, 1.5 and 3 h after SSB infestation (e),

and levels of SA in leaf sheaths of hpl3-1 and WT

plants at 0, 8 and 48 h after BPH infestation (f).

Values are means � SE and were obtained in

one experiment with five biological replicates,

and similar results were obtained in two inde-

pendent experiments. Letters indicate significant

differences among treatments (P < 0.05; Dun-

can’s multiple range test).



(Figure 6f,g) exhibited less preference for feeding on trans-

genic plants over-expressing OsHPL3 (OE-OsHPL3) (Fig-

ure S6), and laid significantly fewer eggs on OE-OsHPL3

plants compared to WT plants (Figure 6f,g, insets). These

results indicate that OsHPL3 positively modulates rice

resistance to BPH.

We have shown that the hpl3-1 mutant had significantly

higher JA and lower GLV levels than the WT (Figure 4). To

determine whether the improved performance of BPH on the

hpl3-1 plants resulted from the decreased GLV levels, we

performed GLV treatment experiments as previously

described (Qi et al., 2011). When the hpl3-1 plants were

individually treated with 125 nmol (Z)-3-hexen-1-ol, BPH

female adults preferred to settle and oviposit on the WT

plants, whereas exogenous application of (Z)-3-hexenal on

the hpl3-1 plants did not exhibit this repellent effect

(Figures 6h and S7). These results suggest that (Z)-3-hex-

en-1-ol supplementation at least partially restored the

phenotype of hpl3-1 plants to the feeding and oviposition

preference of BPH female adults.

Mutation in OsHPL3 increases the attractiveness of a BPH

egg parasitoid and improves its performance

Our previous study showed that other plant volatiles also

play roles in rice defense, including attracting natural ene-

mies of BPH (Lou et al., 2005). To investigate whether the

increase in BPH-induced volatiles in the hpl3-1 plants (Fig-

ures 5b and S5) modulates rice indirect defense by affecting

the attractiveness of a BPH egg parasitoid, the hpl3-1 plants

were infected with BPH and the attractiveness of the BPH

parasitoid A. nilaparvatae was assessed. Compared with

infested WT plants, infested hpl3-1 plants were significantly

more attractive to the parasitoid (Figure 7a). Moreover, the

parasitoid seemed to prefer to parasitize BPH eggs on the

hpl3-1 mutant than on the WT plants, as, in a two-choice

experiment, parasitism of BPH eggs by the wasps was

Figure 5. Levels of TrypPIs and other volatiles and rice resistance to SSB.

(a) Levels of TrypPIs in stems of hpl3-1 and WT plants at 1, 3 and 5 days after infestation by a third-instar SSB larva (non-infestation as control). Values are

means � SE, and were obtained in one experiment with five biological replicates, and similar results were obtained in two independent experiments. Letters

indicate significant differences among lines at each time point (P < 0.05, Duncan’s multiple range test).

(b) Levels of other volatile chemicals. Volatiles were obtained by headspace collections from BPH-infested and non-infested hpl3-1 and WT plants (for 24 h), and

detected as previously described (Lou et al., 2005). Volatiles emitted from individual plants (one per pot) infested with 15 BPH adults for 24 h (BPH) or not infested

(control) were collected and analyzed with five biological replicates. Numbers represent chemicals as follows: (1) 2-heptanone; (2) 2-heptanol; (3) a-pinene; (4)

myrcene; (5) (+)-limonene; (6) (E)-linalool oxide; (7) methyl benzoate; (8) linalool; (9) methyl salicylate; (10) a-copaene; (11) sesquithujene; (12) a-cedrene; (13) (E)-

b-caryophyllene; (14) (E)-a-bergamotene; (15) sesquisabinene A; (16) (E)-b-farnesene; (17) a-curcumene; (18) zingiberene; (19) b-bisabolene; (20) b-sesquiphelland-

rene; (21) (E)-c-bisabolene. Values are percentages of the peak area of the internal standard (IS) (mean � SE). Asterisks indicate significant differences between BPH-

infested WT and hpl3-1 plants (*P < 0.05; Student’s t test). The results shown were obtained in one experiment with five biological replicates, and similar results

were obtained in two independent experiments.

(c) Mass of individual SSB larva 13 days after they were placed on hpl3-1 and WT plants. Inset, death rate of these SSB larva on the hpl3-1 and WT plants. Results

shown (means � SE) were obtained in one experiment with 40 biological replicates for WT, 25 biological replicates for hpl3-1, and similar results were obtained in

two independent experiments. Asterisks indicate significant differences in hpl3-1 compared to WT plants (*P < 0.05, **P < 0.01; Student’s t test).



significantly higher (1.8-fold) on hpl3-1 than WT plants

(Figure 7c). Interestingly, although there was little difference

in female ratio and number of eggs per female adult or

development duration of the wasps that emerged from BPH

eggs on hpl3-1 and the WT plants (Figure S8), the offspring

of wasps on hpl3-1 plants exhibited only 28.54% of mortality

of offspring of the wasps on WT plants (a statistically sig-

nificant difference) (Figure 7b). These results suggest that

loss-of-function of OsHPL3 also enhances the indirect de-

fense response in rice.

OsHPL3 modulates disease resistance probably through

modulating the SA and JA pathways

In addition to increased JA accumulation (Figure 4c,d), we

also found that the SA level was constitutively higher in

stems and leaf sheaths of hpl3-1 plants than in WT plants

(Figure 4e,f). Consistent with the increased SA and JA levels,

the expression levels of the pathogenesis-related (PR) genes

OsPR1a and PR10b were markedly elevated in the hpl3-1

plants (Figure 8a). Previous studies have shown that both

the SA and JA pathways are involved in rice resistance to

bacterial blight cause by Xanthomonas oryzae pv. oryzae

(Xoo) (Mei et al., 2006; Yuan et al., 2007; Yang et al., 2008).

Therefore, we assayed Xoo resistance of the hpl3-1 plants.

Compared with WT plants, the hpl3-1 plants were signifi-

cantly more resistant to Xoo (Figure 8b,c). These results

indicate that the OsHPL3-mediated oxylipin metabolic

pathway is also involved in the rice defense response

against pathogens, probably by synchronously augmenting

JA and SA signaling. Similar synchronously augmented SA

and JA levels were also observed in as-pld rice plants (Qi

et al., 2011). However, the mechanism of how SA signaling

is activated in the mutant remains unknown.

DISCUSSION

In this study, we report that the rice OsHPL3 gene plays

critical roles in modulating plant defense responses. OsHPL3

has intrinsic HPL activity, metabolizing hydroperoxylino-

lenic acid to produce GLVs in planta. Loss of OsHPL3 func-

tion results in increased resistance to the chewing herbivore

SSB but enhanced susceptibility to the phloem-feeding

herbivore BPH. Interestingly, the hpl3-1 plants also exhibited

increased attractiveness to the BPH parasitoid A. nilaparva-

tae. These data strongly suggest that, as an HPL, OsHPL3 is

an important player in modulating rice direct and indirect

defense responses specifically against different invaders by

Figure 6. BPH performance on hpl3-1, WT and

OE-OsHPL3 plants.

(a) Developmental duration of BPH eggs on hpl3-

1 and WT plants. Values are means � SE (n = 5).

Inset, amounts of honeydew per day excreted by

a BPH female adult (FA) feeding on hpl3-1 and

WT plants.

(b–d) Number of BPH nymphs per plant in two-

choice assays on hpl3-1 plants versus WT plants

(b), and OE-OsHPL3 line OE-127 (c) or OE-128 (d)

plants versus WT plants over a 48 h time course

of BPH nymph release. Five replicated plant pairs

were exposed to 15 insects.

(e–g) Number of BPH female adults per plant in

two-choice assays on hpl3-1 plants versus WT

plants (e), and OE-OsHPL3 line OE-127 (f) or OE-

128 (g) plants versus WT plants. Numbers were

counted over a 48 h time course after pairs of

plants were exposed to 15 female adults. Insets,

percentages of BPH eggs on pairs of plants 48 h

after the release of BPH female adults.

(h) Mean percentage of BPH eggs per plant on

pairs of plants: hpl3-1 plants plus 125 nmol of

(Z)-3-hexenal in 10 ll of lanolin versus WT plants

with lanolin only, and hpl3-1 plants plus

125 nmol of (Z)-3-hexen-1-ol in 10 ll of lanolin

versus WT plants with lanolin only, 48 h after the

release of BPH female adults.

Values are means � SE, and were obtained in

one experiment with five biological replicates [a–

h; inset in (a) with 20 biological replicates], and

similar results were obtained in two independent

experiments. Asterisks indicate significant differ-

ences between mutant and WT plants (*P < 0.05,

**P < 0.01; Student’s t test).



modulating the oxylipin pathway. Our studies also provide

compelling evidence that plants may modulate plant–insect

interactions up to the third trophic level by regulating a

single gene.

A previous study showed that HPLs influence the biosyn-

thesis of JA by competitively utilizing a common substrate

with the AOS cascade (Halitschke et al., 2004). Interestingly,

we found that the transcript level of AOS2 was significantly

higher in hpl3-1 than in the WT (Figure S4), suggesting that

OsHPL3 may modulate the production of JA in at least two

ways: by consuming the common substrate and by affecting

the expression of AOS2. OsHPL3 is wound-inducible and

exclusively expressed in leaves (Figure 2) (Chehab et al.,

2006). In contrast, OsHPL1 and OsHPL2 are not wound-

induced (Chehab et al., 2006). Furthermore, the levels of

(Z)-3-hexenal and (Z)-3-hexen-1-ol in the wounded leaves

were markedly different between the WT and the hpl3-1

mutant, which has decreased HPL3 activity toward 13-HPOT

(Figures 3b,c and S3). In addition, stress-induced generation

of aldehydes in rice leaves may be primarily due to the

activity of OsHPL3 (Chehab et al., 2006). These results

together suggest that OsHPL3 is the major wound-induced

HPL in rice leaves.

We showed that mutation in OsHPL3 creates plants with

increased resistance to SSB caterpillars (Figure 5c), whereas

BPH preferred to feed and oviposit on hpl3-1 plants

(Figure 6b,e). The increased SSB resistance of hpl3-1 plants

is most likely due to the elevated levels of SSB-induced JA

and TrypPIs, as our previous findings indicate that JA

signaling and associated TrypPIs play an important role in

rice resistance to SSB (Zhou et al., 2009; Lu et al., 2011).

Furthermore, our previous studies revealed that a high H2O2

level could cause hypersensitive response-like cell death

and thus reduced BPH feeding (Zhou et al., 2009), and that

high levels of (Z)-3-hexen-1-ol also reduced BPH feeding and

oviposition (Qi et al., 2011). Therefore, high levels of (Z)-3-

hexen-1-ol and H2O2 (probably associated with high SA

levels) may constitute the chemical mechanisms of rice

resistance to BPH. Given that the level of (Z)-3-hexen-1-ol but

not BPH-induced H2O2 was significantly reduced in the hpl3-

1 plants (Figures 4b and S9) and the reduced resistance of

Figure 7. Effect of hpl3-1 plants on behavior and performance of Anagrus

nilaparvatae.

(a) Parasitism rates of BPH eggs by A. nilaparvatae in two-choice assays on

hpl3-1 plants versus WT plants. Values are means � SE (n = 9).

(b) Mortality of A. nilaparvatae parasitizing in BPH eggs on hpl3-1 plants and

WT plants. Values are means � SE (n = 9).

(c) Number of A. nilaparvatae attracted by volatiles released from pairs of

plants: hpl3-1 plants versus WT plants, and BPH-infested hpl3-1 plants versus

BPH-infested WT plants. Plants were infested by BPH for 24 h before

A. nilaparvatae release (n = 48).

Results shown were obtained in one experiment with n biological replicates,

and similar results were obtained in two independent experiments. Asterisks

indicate significant differences between hpl3-1 and WT plants [*P < 0.05,

**P < 0.01; Student’s t test in (a) and (b); v2 test for (c)]. ns, no significant

difference.

Figure 8. Rice resistance to bacterial blight.

(a) Enhanced expression of the genes PR1a and PR10b in hpl3-1, as detected

by RT-PCR analysis. OsUBI was used as a loading control. Similar results were

obtained in two independent experiments.

(b) Disease symptoms on two rice bacterial blight (Xoo) strains Xoo 99A (P6)

and DY89031 (K1). Photos were taken at 15 days after inoculation.

(c) Lesion lengths on leaves of hpl3-1 and WT plants at 15 days after

inoculation. Values are means � SE, and were obtained in one experiment

with 30 biological replicates, and similar results were obtained in three

independent experiments. Asterisks indicate significant differences in hpl3-1

plants compared to WT plants (**P < 0.01; Student’s t test).



hpl3-1 plants to BPH was restored by exogenous application

of (Z)-3-hexen-1-ol (Figures 6h and S7), it is possible that the

reduction of BPH resistance in hpl3-1 plants results from the

reduced (Z)-3-hexen-1-ol levels.

Herbivore-induced plant volatiles can attract natural ene-

mies of the herbivores, thereby indirectly protecting plants

(Kessler, 2004; Frost et al., 2008; Dicke, 2009). Our chemical

profile analysis revealed that the hpl3-1 plants released

significantly elevated levels of BPH-induced volatiles except

for GLVs (Figures 5b and S5). The hpl3-1 plants also showed

increased attractiveness to the egg parasitoid A. nilaparva-

tae (Figure 7a,c). JA plays an important role in the produc-

tion of herbivore-induced volatiles in rice (Lou et al., 2005; Qi

et al., 2011). Although the basal JA level was markedly

increased, the BPH-elicited JA level was not enhanced in the

hpl3-1 plants (Figure 4d). A possible explanation for the

increased levels of BPH-elicited volatiles from the hpl3-1

plants is that high basal JA levels in hpl3-1 efficiently trigger

release of volatiles upon BPH attack.

It is well documented that host plants may influence the

survival rate, fecundity, female ratio and developmental

duration of parasitoid progenies directly and/or indirectly by

affecting herbivore egg quality and/or size (Harvey, 2000;

Spitzen and Van, 2005; Onagbola et al., 2007). In rice,

different varieties show distinct performance of the parasit-

oid A. nilaparvatae by affecting the quality or size of BPH

eggs (Lou and Cheng, 1996). Apart from its direct effect on

attractiveness to the egg parasitoid A. nilaparvatae, we

found that mutation in OsHPL3 also influenced the perfor-

mance of A. nilaparvatae. In addition, offspring of the

parasitoid parasitizing BPH eggs on the hpl3-1 plants had a

significantly higher survival rate than those on WT plants

(Figure 7b), although the sizes of BPH eggs did not differ

between the hpl3-1 and WT plants, as they were all

deposited by the same six gravid BPH females over 1 day.

Therefore, the difference in the survival rate of the parasitoid

between the hpl3-1 and WT plants may be attributed to

different chemical surrounding, which may influence the

parasitoid directly or indirectly by affecting the quality of

BPH eggs. The BPH eggs developed significantly faster on

hpl3-1 plants than on WT plants (Figure 6a), suggesting that

hpl3-1 plants are more suitable for BPH egg development,

thereby improving the performance of the egg parasitoid.

Given the obvious repellent role of (Z)-3-hexen-1-ol on BPH

feeding and oviposition, we propose that (Z)-3-hexen-1-ol

may be one of the chemicals that influences BPH egg

development and the survival rate of the parasitoid proge-

nies.

Interestingly, mutation in OsHPL3 also creates plants with

enhanced resistance to bacterial blight and enhanced

expression of PR genes (Figure 8). The increased disease

resistance in the hpl3-1 plants probably resulted from

enhanced JA signaling, as JA-mediated defense is involved

in rice disease resistance and cell death (Lee et al., 2001; Mei

et al., 2006; Yang et al., 2008), as well as the augmented SA

signaling pathway, which has been recognized to play a role

in rice Xoo resistance. Similar synergistic activation of the

SA and JA/ethylene signaling pathways was also observed

in the Arabidopsis lesion-mimic hrl1 mutant (Devadas et al.,

2002). Therefore, certain signaling components of the JA

and SA pathways may fine-tune the overlapping activation

of both SA- and JA-dependent defense responses to confer

resistance against herbivores and pathogens. Identification

of such key components will provide deep insight into the

regulatory network that orchestrates rice defense responses

against diverse herbivores and pathogens.

EXPERIMENTAL PROCEDURES

Plant materials and growth

The recessive mutant hpl3-1 was isolated from an M2 population ofZhonghua 11 (ZH11) (japonica) mutagenized with c-rays (Zhu et al.,2003). The mutant was crossed with an indica rice (Zhenshan 97) togenerate an F2 mapping population. Plants were cultivated in anexperimental field under natural growing conditions for morpho-logical and physiological analysis. Transgenic rice plants weregrown in the phytotron under 12 h light (28 � 2�C)/12 h dark(25 � 2�C) and 70–85% relative humidity.

Map-based cloning, plasmid construction and plant trans-

formation

Using a series of simple sequence repeat and sequence-tagged sitemarkers (http://www.gramene.org/microsat/ssr.html; Table S1), themutant locus was mapped to a 28 kb region on chromosome 2between markers T4 and T6 using 1209 recombinants. The genomicfragments of candidate genes were PCR-amplified, sequenced andcompared with the wild-type sequence for mutation detection. Forcomplementation of the hpl3-1 mutant, a 4.68 kb genomic DNAfragment bearing the OsHPL3 coding region plus 2 kb promoter and1 kb 3¢ end was released from the Nipponbare BAC OSJNBa0089F07and cloned into expression vector pCAMBIA1300 (accession num-ber AF234269) to produce the construct p1300-OsHPL3. This con-struct and the empty vector were then introduced into hpl3-1 calli byAgrobacterium tumefaciens-mediated transformation. More than55 independent transgenic lines were produced, and all showedwild type-like phenotype. To generate OsHPL3 over-expressionlines (OE-OsHPL3), the 1.46 kb OsHPL3 full-length cDNA was clonedinto the plant expression vector p35S-C1301, and introduced intoWT Nipponbare. More than 70 independent OE-OsHPL3 lines wereobtained, and T1 and T2 generation plants were used for all assays.

Insect maintenance and herbivore experiment

Colonies of SSB and BPH were maintained on rice seedlings (cv.Xiushui 11) as described previously (Zhou et al., 2009). A. nila-parvatae Pang et Wang colonies were obtained from rice fields inHangzhou, China, as described previously (Xiang et al., 2008).Freshly hatched SSB larvae were allowed to infest stems (10 larvaeper plant) using five biological replicates for statistical analysis.Larval mass (to an accuracy of 0.1 mg) was measured 13 days afterinfestation. To determine the colonization and oviposition prefer-ence of BPH, pots with a pair of plants (one WT plant and one hpl3-1or OE plant) were individually confined within plastic cages, intowhich 15 gravid adult BPH females or nymphs were introduced,with five biological replicates. The number of BPH on each plant was



calculated at the indicated times after release of BPH. The number ofeggs on each plant was counted under a microscope.

For BPH feeding, a newly emerging macropterous female BPHadult was placed into a small plastic bag (6 · 5 cm), and then fixedon the plant stem, with each plant receiving two females, with 20biological replicates for statistical analysis. The amount of honey-dew excreted by a female adult was weighed (to an accuracy of0.1 mg) 24 h after BPH feeding. To test the survival and develop-mental duration of BPH eggs, plants were individually infested withfive gravid BPH females for 24 h. Newly hatched BPH nymphs fromeach plant were recorded every day. The survival rate and devel-opmental duration of BPH eggs were statistically calculated withfive biological replicates.

Parasitoid behavior and performance bioassay

Behaviors of A. nilaparvatae females in response to rice volatileswere measured in a Y-tube olfactometer, as described previously(Lou et al., 2005). Plants were randomly assigned to BPH andnon-infested treatments. The behavioral response of the parasitoidexposed to the following pairs of odor sources was observed:BPH-infested plants of hpl3-1 versus BPH-infested WT plants(infestation for 24 h) and non-infested plants of hpl3-1 versus non-infested WT plants. For each treatment, ten pairs of plants wereused, and the odor sources were replaced by a new set of 10 plantsafter testing eight wasps. For each odor source combination, a totalof 48 females were tested.

To determine the preference of A. nilaparvatae females for BPHeggs and the performance of the parasitoid on hpl3-1 and WTplants, BPH eggs were laid on two plants (one WT plant and onehpl3-1 plant) in one pot by six gravid females for 1 day, and theneight wasps (six females and two males) were introduced into thepot after remove of the fixed BPH females. The wasps were removed24 h later. The numbers of newly hatched BPH nymphs and newlyemerged wasps (male and female) were recorded for 5 days. Theeggs of the female wasps were examined as previously described(Lou and Cheng, 1996). The parasitism of BPH eggs, the survivalrate, fecundity, female ratio and developmental duration wereevaluated statistically using nine biological replicates.

RNA preparation and RT-PCR analysis

For gene induction, plants were infested with SSB or BPH. Formechanical wounding, plants were individually damaged with 200holes using a needle on the lower part of the rice stems (approxi-mately 2 cm long). JA and SA treatments were as described by Zhouet al. (2009). Plants were individually sprayed with JA (100 mg ml)1)or SA (70 mg ml)1) in 50 mM sodium phosphate buffer. Total RNAwas extracted by using TRIZOL reagent (Invitrogen, Carlsbad, CA,USA) and treated with DNase I using a DNA-free kit (Ambion, http://www.invitrogen.com/site/us/en/home/brands/ambion.html). First-strand cDNA was synthesized by Superscript III reverse transcrip-tase according to the manufacturer’s instructions (Invitrogen). TheOsHPL3, OsPR1 and OsPR10 transcripts were measured by RT-PCR.The expression levels of OsHPL3 in different tissues and stages andin transgenic plants, and of AOS2 and JAmyb, were determinedusing real-time PCR (Roche, http://www.roche.com/index.htm).Primer sequences are listed in Table S1.

HPL enzyme assay

The OsHPL3 protein was expressed in yeast as previously described(Zhu et al., 2006). The microsomal fraction was prepared from col-lected yeast cells and was suspended in Milli-Q water (Millipore,Billerica, MA, USA). Substrates were incubated with 6 lg micro-somal proteins in a 500 ll volume with or without NADH and alco-

hol dehydrogenase (Duan et al., 2005; Chehab et al., 2006). Thereaction mixture was further monitored using a DU 800 UV-visiblespectrophotometer (Beckman, https://www.beckmancoulter.com/).To assay leaf HPL activity, proteins were partially purified from WTand hpl3-1 leaves as described by Duan et al. (2005). In brief, HPLactivity assays were performed with 0.1 mM NADH and 50 units/mlyeast alcohol dehydrogenase. The oxidation of NADH was moni-tored using a DU 800 UV-visible spectrophotometer by followingthe decrease in absorbance at 340 nm, with background oxidationof NADH without the substrates as a control.

Western blot analysis

An anti-OsHPL3 peptide (CGTSFTKLDKRELTPS) antibody wasraised and purified by antigen affinity purification. Total proteinswere extracted from leaves using a buffer containing 50 mM Tris/HCl (pH 6.8), 4.5% SDS, 7.5% b-mercaptoethanol and 9 M urea. ForWestern blot analysis, proteins (30 lg) were separated on 10% SDS–PAGE and electrophoretically transferred to nitrocellulose mem-branes (Millipore). Immunoblot analysis was performed with theanti-OsHPL3 antibody.

JA, GLV and SA detection and GLV treatment

Plants (one per pot) were randomly assigned to SSB, BPH andcontrol treatments. Stems of WT, hpl3-1 and OE-OsHPL3 wereharvested at 0, 1.5 and 3 h after SSB feeding, as JA reaches peaklevels at approximately 3 h after SSB feeding (Zhou et al., 2011).Leaf sheaths were harvested 0, 8 and 48 h after BPH feeding. JA andSA levels were analyzed by GC-MS using labeled internal standardsas described by Lou and Baldwin (2003). Each treatment at eachtime interval was biologically replicated five times. GLV emissions[(Z)-3-hexenal and (Z)-3-hexen-1-ol] were analyzed with a gas ana-lyzer (zNoseTM 4200, Electronic Sensor Technology, http://www.estcal.com/) using the same method as described by Zhouet al. (2009). (Z)-3-hexenal and (Z)-3-hexen-1-ol concentrations wereexpressed as peak area per mg fresh leaves. Five biological repli-cates were used for each genotype (hpl3-1 and WT). For GLVtreatment, plants were individually treated with 125 nmol (Z)-3-hexenal or (Z)-3-hexen-1-ol in lanolin paste on stems. Control plantsreceived the same volume of lanolin paste only, as previouslydescribed (Qi et al., 2011).

Collection, isolation and identification of other rice volatiles

Collection, isolation and identification of rice volatiles were performedas previously described (Lou et al., 2005). Volatiles emitted from indi-vidual plants (one per pot) infested with 15BPH adults for24 h (BPH) ornon-infested (control) were collected, with five biological replicates foreach treatment. The amounts of compounds are expressed as per-centages of peak areas relative to the internal standard per 8 h oftrapping one plant (for details, please see Appendix S1).

TrypPI analysis and quantification of H2O2

Plants from each line were randomly assigned to SSB and controltreatment. Plants were infested by SSB third-instar larvae (one larvaper plant) for 1, 3 and 5 days, and then stems (0.12–0.15 g persample) were harvested. TrypPI concentrations were measuredusing a radial diffusion assay as described by van Dam et al. (2001).Each treatment was performed with five biological replicates. ForH2O2 quantification, WT and hpl3-1 plants were randomly assignedto BPH and non-infested control groups. Leaf sheaths were har-vested at 0, 3, 8 and 24 h after treatment, with five biological repli-cates. H2O2 concentrations were then determined as described byLou and Baldwin (2006).



Pathogen inoculation

Rice plants were planted in an isolated paddy field. Eight-week-oldplants were inoculated with Philippine race P6 (PXO99A) and Korearace K1 (DY89031) as previously described (Yuan et al., 2007). Lesionlength was recorded at 15 days post-inoculation. Thirty leaves fromten plants (three leaves per plant) were used for statistical analysis.

Data analysis

Differences in herbivore-induced JA, SA, H2O2, TrypPIs and GLVswere analyzed by one-way ANOVA. If the ANOVA was significant(P < 0.05), Duncan’s multiple range test was used to detect signifi-cant differences between groups. Differences in the attractivenessof A. nilaparvatae to BPH between lines were tested by v2 test. Dif-ferences in experiments with two treatments were determined byStudent’s t test.

ACKNOWLEDEGMENTS

We are grateful to Jianjun Wang and Xiaoming Zhang (ZhejiangAcademy of Agricultural Sciences, China) for help with rice growthand maintenance, and Xiaofeng Cui for critical reading. This re-search was supported by grants from the National Research Pro-gram of China (2011CB100700) and the National Natural ScienceFoundation of China (91117018 and 30730064) and by the ChineseAcademy of Sciences.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article.Figure S1. Light-induced lesions and agronomic traits of hpl3-1.Figure S2. Alignment of the amino acid sequences of SLM1,OsHPL1, OsHPL2 and Arabidopsis HPL.Figure S3. In vitro enzyme activity assays of recombinant OsHPL3using 9-/13- hydroperoxides.Figure S4. Transcript levels of AOS2 and JAmyb.Figure S5. Typical chromatograms of volatile compounds obtainedby headspace collections from BPH-infested (for 24 h) and non-infested plants.Figure S6. Overexpression of OsHPL3 in transgenic lines.Figure S7. Number of BPH female adults on WT and hpl3-1plants.Figure S8. Developmental duration, fecundity and female ratio ofAnagrus nilaparvatae emerged from BPH eggs on hpl3-1 and WTplants.Figure S9. H2O2 concentrations in hpl3-1 and WT plants.Table S1. Primers used for mapping and gene expression analysis.Appendix S1. Experimental procedure.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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