The rice hydroperoxide lyase OsHPL3 functions in defense responses by modulating the oxylipin pathway Xiaohong Tong 1,2,† , Jinfeng Qi 1,† , Xudong Zhu 3,† , Bizeng Mao 1 , Longjun Zeng 2 , Baohui Wang 1 , Qun Li 2 , Guoxin Zhou 1 , Xiaojing Xu 4 , Yonggen Lou 1, * and Zuhua He 1,2, * 1 College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China, 2 National 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, 3 China National Rice Research Institute, 359 Tiyuchang Road, Hangzhou 31006, China, and 4 National 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 (Sta ˚ l)] 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 763 The Plant Journal ª 2012 Blackwell Publishing Ltd The Plant Journal (2012) 71, 763–775 doi: 10.1111/j.1365-313X.2012.05027.x
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The rice hydroperoxide lyase OsHPL3 functions in defenseresponses by modulating the oxylipin pathway
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.
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).
766 Xiaohong Tong et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 763–775
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
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.
Hydroperoxide lyase functions in rice defense 767
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 763–775
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-
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)
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).
Hydroperoxide lyase functions in rice defense 769
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 763–775
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
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
772 Xiaohong Tong et al.
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 763–775
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).
Hydroperoxide lyase functions in rice defense 773
ª 2012 The AuthorsThe Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 763–775
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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