The Chloroplast-Localized Phospholipases D 4and5 Regulate ...The Chloroplast-Localized Phospholipases D a4and5a Regulate Herbivore-Induced Direct and Indirect Defenses in Rice1[C][W]

The Chloroplast-Localized Phospholipases D a4 and a5Regulate Herbivore-Induced Direct and Indirect Defensesin Rice1[C][W]

Jinfeng Qi, Guoxin Zhou, Lijuan Yang, Matthias Erb, Yanhua Lu, Xiaoling Sun,Jiaan Cheng, and Yonggen Lou*

National Key Laboratory of Rice Biology, Institute of Insect Science, Zhejiang University, Hangzhou 310029,China (J.Q., G.Z., L.Y., Y. Lu, J.C., Y. Lou); Department of Molecular Ecology, Max Planck Institute forChemical Ecology, 07745 Jena, Germany (M.E.); and Tea Research Institute, Chinese Academy of AgriculturalScience, Hangzhou 310008, China (X.S.)

The oxylipin pathway is of central importance for plant defensive responses. Yet, the first step of the pathway, the liberation oflinolenic acid following induction, is poorly understood. Phospholipases D (PLDs) have been hypothesized to mediate thisprocess, but data from Arabidopsis (Arabidopsis thaliana) regarding the role of PLDs in plant resistance have remainedcontroversial. Here, we cloned two chloroplast-localized PLD genes from rice (Oryza sativa), OsPLDa4 and OsPLDa5, both ofwhich were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis, mechanical wounding,and treatment with jasmonic acid (JA). Antisense expression of OsPLDa4 and -a5 (as-pld), which resulted in a 50% reduction ofthe expression of the two genes, reduced elicited levels of linolenic acid, JA, green leaf volatiles, and ethylene and attenuatedthe SSB-induced expression of a mitogen-activated protein kinase (OsMPK3), a lipoxygenase (OsHI-LOX), a hydroperoxidelyase (OsHPL3), as well as a 1-aminocyclopropane-1-carboxylic acid synthase (OsACS2). The impaired oxylipin and ethylenesignaling in as-pld plants decreased the levels of herbivore-induced trypsin protease inhibitors and volatiles, improved theperformance of SSB and the rice brown planthopper Nilaparvata lugens, and reduced the attractiveness of plants to a larvalparasitoid of SSB, Apanteles chilonis. The production of trypsin protease inhibitors in as-pld plants could be partially restored byJA, while the resistance to rice brown planthopper and SSB was restored by green leaf volatile application. Our results showthat phospholipases function as important components of herbivore-induced direct and indirect defenses in rice.

Induced plant defenses play an important role inprotecting plants from herbivore attack (Browse andHowe, 2008). Their activation depends on a complexsignaling network with the oxylipin pathway at itscenter (Bostock, 2005; Browse and Howe, 2008; Howeand Jander, 2008). The biosynthesis of oxylipins startswith the release of linolenic acid (LeA) from chlo-roplast membranes. LeA is then oxygenated via 9- or13-lipoxygenase (LOX) into hydroperoxy polyunsatu-rated fatty acids, which are the common substrates forat least seven different enzymes, including allene

oxide synthase and hydroperoxide lyase (HPL; Feussnerand Wasternack, 2002). The metabolites of the alleneoxide synthase and HPL branch of the pathway arejasmonates and green leaf volatiles (GLVs), both ofwhich are important signals in plant defenses (Kesslerand Baldwin, 2001; Shiojiri et al., 2006; Browse andHowe, 2008). At present, the first crucial step inoxylipin biosynthesis and induction, the release ofLeA from chloroplast membranes, is not well under-stood. Plant phospholipases D (PLDs) have receivedspecial attention as possible mediators of this reaction(Wang et al., 2000; Bargmann et al., 2009). PLDs cleavephospholipids into phosphatidic acid (PA) and freehead groups such as choline (Li et al., 2009; Hong et al.,2010). PA may then be converted to LeA (Ryu andWang, 1998; Sang et al., 2001), thereby enabling anincrease in jasmonate production. PLDs may alsostimulate acylhydrolases or phospholipase A activity,which may induce jasmonate signaling (Wang et al.,2000). In accordance with these two hypotheses, anti-sense expression ofAtPLDa1 in Arabidopsis (Arabidopsisthaliana) reduced PA levels, LOX activity, wound-induced JA levels, and wound-induced gene expression(Wang et al., 2000). However, Bargmann et al. (2009)recently demonstrated that plda1 and plda1/d doubleknockouts of Arabidopsis do not show any changes inwound-induced protein kinase activity, AtLOX2 gene

1 This work was supported by the National Basic ResearchProgram of China (grant no. 2010CB126200), the Natural ScienceFoundation of Zhejiang Province (grant no. D3080282), the Innova-tion Research Team Program of the National Natural Science Foun-dation of China (grant no. 31021003), the National Natural ScienceFoundation of China (grant no. 30871644), and an earmarked fundfor the Modern Agro-industry Technology Research System.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www. plantphysiol.org) is:Yonggen Lou ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.111.183749

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expression, JA biosynthesis, and resistance against theherbivore Pieris rapae. The authors attributed this sur-prising difference to the possibility that the antisenseconstructs used by Wang et al. (2000) may have sup-pressed other PLD isoforms in Arabidopsis.

PLDs are a large family of heterologous enzymes witha variety of functions. It has been reported that PLDs andtheir main product, PA, because of their involvementin membrane degradation, vesicular transport, mem-brane tethering, signal transduction, and hormoneproduction or action, play important roles in plantgrowth, development, and response to various abioticand biotic stresses, including wounding and pathogeninfection (Choi et al., 2005; Wang et al., 2006; Honget al., 2008, 2009, 2010; Wan et al., 2009; Yamaguchiet al., 2009). Different PLDs have distinct, but some-times overlapping, functions (Li et al., 2009). In Arab-idopsis, for example, both PLDa1 and -a3 enhanceplant osmotic stress responses, but they act throughdifferent mechanisms: PLDa1 mediates the abscisicacid effect on stomatal movement to reduce water loss,whereas PLDa3 promotes root growth (Hong et al.,2010). Several PLDs have been reported to be involvedin plant defense responses. In rice (Oryza sativa),silencing OsPLDb1 caused hypersensitive response-like cell death, phytoalexin production, and increasedplant resistance to pathogens (Yamaguchi, et al., 2009).Krinke et al. (2009) found that PLD activation is anearly component of the salicylic acid (SA) signalingpathway in Arabidopsis. PLDs in soybean (Glycinemax) activated a wound-activated mitogen-activatedprotein kinase (MAPK; Lee et al., 2001), a homolog ofwhich is responsible for the production of wound-

induced JA, SA, and ethylene in Nicotiana attenuata(Wu et al., 2007). AtPLDa1 finally positively regulatedreactive oxygen species generation and was involvedin ethylene signaling in Arabidopsis (Fan et al., 1997; Liet al., 2009; Zhang et al., 2009). In summary, while PLDsseem to be central to many plant regulatory processes,their role in the wound response remains unclear. Thisis especially evident for PLDs in monocots, wherenothing is known about their involvement in oxylipin-mediated defenses against herbivores.

Therefore, we isolated two herbivore-induced ricePLD genes, OsPLDa4 and OsPLDa5, both of whichlocalize in chloroplasts (McGee et al., 2003) and share90% sequence identity. Using a reverse genetics ap-proach, we obtained rice lines with reduced expressionofOsPLDa4 andOsPLDa5 (as-pld). We determined therole of these two genes in herbivore-induced defenseby measuring the expression of several oxylipin andethylene biosynthesis-related genes, concentrationsof phytohormones, as well as the direct and indirectresistance against a chewing herbivore, the stripedstem borer (SSB [Chilo suppressalis]), and a phloemfeeder, the rice brown planthopper (BPH [Nilaparvatalugens]). Our results provide evidence for the involve-ment of OsPLDa4 and -a5 in rice herbivore defense.

RESULTS

Isolation and Characterization of Rice OsPLDa4 and -a5

By reverse transcription (RT)-PCR, we cloned thefull-length cDNAs of two SSB-induced OsPLD genes.

Figure 1. Mean expression levels (relativeto expression levels of OsACT; +SE; n = 5)of OsPLDa4 and -a5 in rice stems afterdifferent treatments. A and D, SSB. B andE, Mechanical wounding (W). C and F, JAand sodium phosphate buffer (BUF). As-terisks indicate significant differences be-tween treatments and controls (0-h timepoint for A, B, D, and E; buffer for C and F;* P , 0.05, ** P , 0.01, Student’s t test).

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The two genes included open reading frames of 2,499and 2,529 bp, which encode 832 amino acids with apredicted molecular mass of 92.39 kD and a pI of 6.183and 842 amino acids with a calculated molecular massof 93.19 kD and a theoretical pI of 6.148, respectively(Supplemental Figs. S1 and S2). Sequence alignmentrevealed high similarities (99% and 99%) of the twoPLD genes to those of previously identified rice PLDgenes (AK100278 and AK119861; Supplemental Fig.S1), suggesting that the two cloned OsPLD genes areOsPLDa4 (AK100278) and OsPLDa5 (AK119861;Yamaguchi et al., 2009). Moreover, both of the two PLDgenes share 90% identity in nucleotide sequence and84% identity in amino acid sequence (SupplementalFig. S2).Quantitative (q)RT-PCR analysis of rice stems re-

vealed low constitutive expression of OsPLDa4 and-a5, while SSB caterpillar attack, mechanical wound-ing, and JA treatment resulted in a rapid increase intranscript levels in infested or treated stems (Fig. 1).In contrast, five other tested rice PLDs, OsPLDa1,OsPLDa2, OsPLDa3, OsPLDh1, and OsPLDh2, didnot change their expression profile after SSB infesta-tion (Supplemental Fig. S6, A–E). BPH infestation orSA treatment did not induce transcript accumulationof OsPLDa4 and -a5, except for the 24-h harvest ofBPH infestation, when transcript levels of OsPLDa5in BPH treatment were higher than in noninfestedtreatment (Supplemental Fig. S3). Hence, OsPLDa4and -a5 share similar induced expression profiles andmay be involved in JA-related insect responses inrice.

Antisense Expression of OsPLDa4 and -a5 Reduces

Elicited Levels of LeA as Well as OsMPK3 mRNA ButNot Hydrogen Peroxide

As OsPLDa4 and -a5 share high sequence identity,similar induced expression profiling, and chloroplastlocalization, it is likely that they are functional ho-mologs. Therefore, we cosilenced the two genes byAgrobacterium tumefaciens-based transformation (Sup-plemental Fig. S4) and obtained three independentlytransformed lines (L10-2, L10-6, and L10-22) with asingle insertion (Supplemental Fig. S5). qRT-PCRshowed that expression of OsPLDa4 and -a5 in SSB-infested stems decreased markedly in two of the as-pldlines compared with identically treated wild-typeplants; the levels of OsPLDa4 and -a5 mRNA in thetwo lines L10-2 and L10-6 were 38.92% to 59.25% and47.99% to 61.46%, respectively, of those in wild-typeplants 1 and 2 h after SSB infestation (Fig. 2A). TheOsPLDa4 and -a5 antisense constructs did not cosilencethe transcript accumulation of OsPLDa1, OsPLDa2,OsPLDa3, OsPLDh1, and OsPLDh2 (Supplemental Fig.S6, A–E), all of which share 66% to 77% sequencesimilarity with OsPLDa4 (Supplemental Fig. S6, A–E).There was no obvious difference in growth phenotypebetween wild-type plants and as-pld lines during theirwhole development (Supplemental Fig. S5). For most of

the following experiments, we compared two as-pldlines (L10-2 and L10-6) with untransformed plants.

The maximal accumulation of LeA occurred 15 minafter SSB infestation (Fig. 2B). Constitutive and SSB-induced LeA levels (15 min after infestation) in twoas-pld lines were lower than those in wild-type plants(Fig. 2B). Antisense expression of OsPLDa4 and -a5had no influence on the SSB-induced expression levelsof OsMPK6 (Supplemental Fig. S6F) but significantlyreduced the expression of OsMPK3 less than 1 h afterSSB infestation (Fig. 2C). Basal hydrogen peroxide(H2O2) levels were significantly higher in wild-typeplants than in as-pld lines. However, after BPH infes-tation for 3, 8, and 24 h, there was no difference in

Figure 2. Mean expression levels (relative to expression levels ofOsACT; +SE; n = 5) of OsPLDa4 and -a5 (A) and OSMPK3 (C) and LeAlevels (+SE; n = 5; B) in wild-type plants (WT) and as-pld lines atdifferent time points after SSB infestation. Letters indicate significantdifferences between lines within one time point (P , 0.05, Duncan’smultiple range test). Asterisks indicate significant differences betweentreatments and controls (0-h time point) within one line (* P , 0.05,** P , 0.01, Student’s t test). FW, Fresh weight.

Role of OsPLDa4 and OsPLDa5 in Rice Defense Responses

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H2O2 levels between as-pld lines and wild-type plants(Supplemental Fig. S7).

OsPLDa4 and -a5 Influence JA, SA, Ethylene, and

GLV Biosynthesis

Phytohormone analysis showed that basal JA levelsin the as-pld lines did not differ significantly fromthose of wild-type plants (Fig. 3A). However, JA levelsincreased more strongly in wild-type plants than in as-pld after infestation with SSB (Fig. 3A). Equally, therewas no difference in constitutive SA levels between theas-pld lines and wild-type plants (Fig. 3E), but 3 h afterSSB infestation, SA levels in as-pld lines were signifi-cantly higher than those in wild-type plants (Fig. 3E).

Ethylene accumulation in as-pld plants was less than inwild-type plants 6 to 48 h after SSB infestation (Fig.3C). Consistent with the results of JA and ethylene,antisense expression of OsPLDa4 and -a5 significantlydecreased the transcript levels of OsHI-LOX (startingless than 0.5 h after infestation; Fig. 3B), a 13-lipoxy-genase gene involved in herbivore-induced JA biosyn-thesis in rice (Zhou et al., 2009), and OsACS2 (startingless than 1 h after infestation; Fig. 3D), a 1-amino-cyclopropane-1-carboxylic acid synthase gene that isinvolved in herbivore-induced rice ethylene produc-tion (Lu et al., 2011).

We also analyzed the emission of GLVs in as-pld andwild-type plants. The levels of constitutive (Z)-3-hexenal and (Z)-3-hexen-1-ol from as-pld lines (L10-2

Figure 3. Levels of JA, ethylene, SA,(Z)-3-hexenal, and (Z)-3-hexen-1-oland expression levels of OsHI-LOX, OsACS2, and OSHPL3 inas-pld lines and wild-type plants(WT) elicited by various treatments.A and E, Mean levels (+SE; n = 5) ofJA and SA in stems of three as-pldlines and wild-type plants at 0, 1.5,and 3 h after infestation by SSB. C,Mean levels (+SE; n = 6) of ethylenein two as-pld lines and wild-typeplants that were individually in-fested by a third-instar SSB larva.F and G, Mean levels (+SE) of (Z)-3-hexenal and (Z)-3-hexan-1-ol (peakarea mg21 fresh mass) in leaves oftwo as-pld lines andwild-type plantsbefore (control [Con]; n = 5) andimmediately after leaves were cutinto small pieces (wounding [W];n = 13). B, D, and H, Mean expres-sion levels (relative to expressionlevels of OsACT; +SE; n = 5) ofOsHI-LOX (B), OsACS2 (D), andOSHPL3 (H) in stems of two as-pldlines and wild-type plants at 0,0.5, 1, and 2 h after infestation bySSB. Letters in G and F indicatesignificant differences betweentreatments. In the other graphs, let-ters indicate significant differencesbetween lines within time points(P, 0.05, Duncan’s multiple rangetest). Asterisks indicate significantdifferences between treatmentsand controls (0-h time point) withinone line (* P , 0.05, ** P , 0.01,Student’s t test). FW, Fresh weight.

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and L10-6) did not differ from those from wild-typeplants (Fig. 3, F and G), but the wound-inducedemission of (Z)-3-hexenal and (Z)-3-hexen-1-ol fromas-pld lines was significantly lower than that fromwild-type plants (Fig. 3, F and G). The reduction inexpression levels of OsHPL3, a wound-induced hydro-peroxide lyase that exclusively catalyzes the cleavageof 13-hydroperoxy LeA into GLVs (Chehab et al., 2006),was also reduced in as-pld lines, albeit somewhat later,at 1 and 2 h after herbivore infestation (Fig. 3H).


Elicited Trypsin Protease Inhibitor Levels

Constitutive trypsin protease inhibitor (TrypPI) levelsin as-pld lines did not differ from those in wild-typeplants (Fig. 4, A and B). However, SSB- or BPH-elicitedTrypPI activity in as-pld lines L10-2 and L10-6, mea-sured 3 d after the start of herbivore infestation, wasreduced compared with wild-type plants (Fig. 4, A andB). Simultaneous JA treatment and SSB infestationinduced higher TrypPI levels in both wild-type andas-pld plants compared with buffer + SSB treatment.However, the TrypPI levels in as-pld plants treated withJA + SSB were still lower than those in identicallytreated wild-type plants (Fig. 4C), suggesting an im-portant but not exclusive role of the oxylipin pathwayin the herbivore-induced production of TrypPI in rice.Treatment by GLVs (Z)-3-hexenal and (Z)-3-hexen-1-olhad no effect on SSB-induced TrypPI levels (Fig. 4D),indicating that the reduced TrypPI levels in as-pld lineswere related to the reduced elicited JA levels ratherthan the reduced GLV levels.


Resistance against SSB and BPH

SSB caterpillars gained more mass on as-pld linesthan onwild-type plants (Fig. 5A). Consistent with thisfinding, as-pld plants were more severely damaged bySSB and survived less than wild-type plants did (Fig.5D). When the different rice genotypes were exposedto a BPH colony, on the other hand, BPH female adultswere more often found on as-pld lines than on wild-type plants (Fig. 6, A and B). Similarly, BPH femaleadults laid significantly more eggs on as-pld lines thanon wild-type plants (Fig. 6C). BPH nymphs also pre-ferred to feed on as-pld lines over wild-type plants(Fig. 6, D and E), and BPH nymphs that fed on the as-pld lines had higher survival rates than those that fedon wild-type plants (Fig. 6F). There were differencesbetween the as-pld lines (L10-2 and L10-6) and wild-type plants in terms of how they tolerated BPH infes-tation: as-pld lines died more rapidly than wild-typeplants when they were infested by BPH female adults(Fig. 6G). Fourteen days after infestation by 12 BPHfemale adults, as-pld plants had completely wilted,whereas only the outer leaf sheaths showed necrosis inwild-type plants (Fig. 6G).

To determine if the improved performance of SSBcould be due to the reduced GLV production and ifthe improved performance of BPH resulted from thedecrease in GLVs or JA, we conducted a series ofcomplementation experiments. Exogenous applica-tion of GLVs [containing (Z)-3-hexenal and (Z)-3-hexen-1-ol, each at 250 nmol per plant] on wild-typeplants reduced the increases in SSB caterpillar mass

Figure 4. Mean TrypPI levels (+SE; n = 5) inas-pld lines and wild-type plants (WT)elicited by various treatments. A, Stemsof two as-pld lines andwild-type plants 3 dafter plants were individually infested by15 female BPH adults or not infested. B,Stems of two as-pld lines and wild-typeplants 3 d after plants were individuallyinfested by a third-instar SSB larva or keptuntreated (control [Con]). C, Stems of twoas-pld lines and wild-type plants that werefirst individually sprayed with 2 mL ofeither JA (100 mg mL21; SSB+JA) or thesodium phosphate buffer (SSB+BUF) for1 d, followed by third-instar SSB larvaeinfestation for 3 d. D, Stems of two as-pldlines and wild-type plants that were firstindividually treated with 250 nmol of (Z)-3-hexenal (SSB+HAL), (Z)-3-hexen-1-ol(SSB+HOL), or lanolin (SSB+LAN), fol-lowed by third-instar SSB larvae infesta-tion for 3 d. Letters indicate significantdifferences among treatments (A–C) oramong lines within the same treatment(D; P , 0.05, Duncan’s multiple rangetest).





(Fig. 5B). For BPH preference, application of JA on as-pld plants did not alter the feeding and ovipositionpreference of BPH female adults for the as-pld lineL10-6 (Figs. 6, B and C, and 7C; Supplemental Fig.S8J), whereas the preference of BPH female adultschanged when GLVs were applied on as-pld plants:BPH females gradually shifted their preference fromas-pld plants to wild-type plants when GLVs wereapplied in increasing doses (from 0 to 500 nmol GLVs;Figs. 6 and 7; Supplemental Fig. S8), suggesting arepellent role of GLVs to BPH. When the as-pld plantswere individually supplemented with 125 or 250 nmolof (Z)-3-hexen-1-ol, BPH female adults preferred tosettle and oviposit on wild-type plants (Fig. 7B; Sup-plemental Fig. S8, D and E). Exogenous application of(Z)-3-hexenal on the as-pld or wild-type plants did nothave this repellent effect (Fig. 7, B and C; SupplementalFig. S8, F–I). Hence, (Z)-3-hexen-1-ol supplementationfully restored the feeding and oviposition preference ofBPH female adults for the as-pld plants. These and our

previous findings suggest that GLVs are involved inrice resistance to SSB and BPH and that OsPLDa4 and-a5 increase the plant’s defense capacity by stimulatingtheir release.

Antisense Expression of OsPLDa4 and -a5 Decreases

Herbivore-Induced Volatiles and Their Attraction of aLarval Parasitoid of SSB

We collected and analyzed the volatiles emittedfrom wild-type and as-pld plants that were infestedby SSB. The results show that SSB infestation signifi-cantly enhanced the release of volatiles in both wild-type and as-pld plants (Fig. 8; Table I). While there wasno difference in constitutive volatile release betweenwild-type plants and as-pld plants, SSB-infested plantsof as-pld lines L10-2 or L10-6 emitted significantlylower amounts of induced volatiles than wild-typeplants. The emission of 14 compounds was signifi-cantly reduced in as-pld lines (Fig. 8; Table I).

Figure 5. Direct and indirect resis-tance of wild-type plants (WT) andas-pld lines to SSB. A and B, Meanincreased mass (%; +SE) of individ-ual 7-d-old SSB larvae fed on wild-type plants and as-pld lines L10-2and L10-6 (A; n = 24) or wild-typeplants that were treated with lano-lin or GLVs (B; n = 30) 7 d after thelarvae were placed on plants. C,Number of A. chilonis attracted byvolatiles released from either non-manipulated plants (control [Con])or plants infested by SSB for 24 h(n = 48): the wild type versus L10-2and the wild type versus L10-6. D,Damaged phenotypes of as-pldlines L10-2 and L10-6 and wild-type plants that were individuallyinfested with a SSB third-instar lar-vae for 9 d. Letters indicate signif-icant differences among lines (A) ortreatments (B; P , 0.05, Duncan’smultiple range test). Asterisks in-dicate significant differences be-tween odor sources (* P , 0.05,** P , 0.01, x2 test).

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Consistent with the decrease in SSB-induced vola-tiles in as-pld lines, the volatiles emitted from infestedas-pld lines were less attractive to Apanteles chilonis, alarval parasitoid of SSB, than those from SSB-infestedwild-type plants (Fig. 5C). This suggests that OsPLDa4and -a5 participates in the indirect defense responsein rice.

DISCUSSION

Here, we present evidence for the involvement oftwo PLDs in the wound- and insect-mediated induc-

tion of the oxylipin and ethylene pathways and indirect and indirect defenses of rice. Several lines ofevidence support the hypothesis that OsPLDa4 and-a5 play an important, specific role in the woundresponse. First, transcripts of OsPLDa4 and -a5 in-crease after infestation with SSB caterpillars, wound-ing, and JA but not after treatment with SA and BPH(Fig. 1; Supplemental Fig. S3). Second, antisense ex-pression of OsPLDa4 and -a5 decreases elicited levelsof JA, ethylene, and GLVs (Fig. 3) but not H2O2(Supplemental Fig. S7). Third, OsPLDa4 and -a5 arelocalized in chloroplasts (McGee et al., 2003), in whichmost steps of JA biosynthesis take place (Feussner and

Figure 6. BPH performance on as-pld lines L10-2 and L10-6 andwild-type (WT) plants and toler-ance to BPH infestation. A, B, D,and E, Mean number (+SE; n = 5–6)of BPH female adults (A and B) ornymphs (D and E) per plant on pairsof plants, L10-2 versus the wildtype and L10-6 versus the wildtype, at different time points. C,Mean percentage (+SE; n = 5–6) ofBPH eggs per plant on pairs ofplants as stated above, 48 h afterthe release of the insects. F, Meansurvival rate (+SE; n = 8) of BPHnymphs on as-pld lines L10-2 andL10-6 and wild-type plants, 6 dafter the release of the insects. G,Damaged phenotypes of as-pldlines L10-2 and L10-6 and wild-type plants after they were infestedby 12 BPH female adults for 14 d.Asterisks indicate significant differ-ences between pairs of lines (A–E;* P, 0.05, ** P, 0.01, Student’s ttest). Letters indicate significant dif-ferences between lines (F; P ,0.05, Duncan’s multiple rangetest).





Wasternack, 2002). Although the role of AtPLDa1 inthe wound-induced JA production in Arabidopsishas recently been debated (Bargmann et al., 2009), theauthors could not exclude that other PLDs playimportant roles in this process. Since the antisensesequence that we used to inhibit expression levels ofPLDa4 and -a5 had no high similarity to other ricegenes except for the ones tested here, we estimate theprobability of cosilencing as very low. Our study thusclearly supports the notion that PLDs can have acentral function in the wound response. Furtherstudies will be required to elucidate if PLDs have adifferent role in monocotyledons or if they are uni-versal triggering points of oxylipin biosynthesis inplants. Furthermore, the identification of insertionmutants of OsPLDa4 and -a5 will make it possible toassess if the two PLDs are indeed functionally redun-dant.

PLDs have been reported to be implicated inethylene signaling (Pinhero et al., 2003; Testerinket al., 2007) and also have long been hypothesized tobe involved in JA production by regulating LeAlevels (Ryu and Wang, 1998), LOX activity (Wanget al., 2000), and MAPK signaling (Lee et al., 2001).Here, we also found that antisense expression ofOsPLDa4 and -a5 decreases elicited levels of GLVs(Fig. 3). Therefore, we determined the changes inLeA concentrations and the expression levels ofOsHI-LOX, OsHPL3, OsACS2, and two MAPK genes,OsMPK3 andOsMPK6, the homologs (NaMAPK3 andNaMAPK6) of which are responsible for the produc-tion of wound-induced JA, SA, and ethylene in N.attenuata (Wu et al., 2007). Our results show thatantisense expression of OsPLDa4 and -a5 did notinfluence the expression levels of OsMPK6 (Supple-mental Fig. S6F) but decreased the levels of inducedLeA and the expression levels of OsHI-LOX,OsMPK3, OsHPL3, and OsACS2 (Figs. 2 and 3). Therelease of GLVs after wounding is transient; thus, thedecreases in levels of GLVs in as-pld lines were not theresult of the reduction of OsHPL transcript levels thatwere significantly lower than those in wild-type plants1 and 2 h after SSB infestation (Fig. 3). Therefore, theinfluence of OsPLDa4 and -a5 on the oxylipin path-way may occur via two routes: first, via influencingLeA, the common substrate for JA and GLV biosyn-thesis; and second, via modulation of LOX activity andMAPK signaling, which subsequently regulate thebiosynthesis of JA and GLVs. The effect of OsPLDa4and -a5 on ethylene biosynthesis may occur mainly viaregulating MAPK signaling, which in turn affects ACSactivity (Lee et al., 2001; Wu et al., 2007). In the future,it will be important to measure actual MAPK activityin addition to gene expression to substantiate thishypothesis.

Antisense expression of OsPLDa4 and -a5 in ricedecreased plant resistance against SSB (Fig. 5, A andD). This coincided with impaired induced levels ofTrypPIs (Fig. 4B) and GLVs (Fig. 3), suggesting that theOsPLDa4- and -a5-mediated signal cascade plays animportant role in rice defenses, including TrypPI andGLV production. Both TrypPIs (Zavala et al., 2004)and GLVs (Vancanneyt et al., 2001; Shiojiri et al., 2006;Chehab et al., 2008) have been implicated in plantdefense against herbivores. In rice, TrypPIs have beenreported to influence the larval performance of SSB(Zhou et al., 2009), and here we found that GLVs [(Z)-3-hexenal and (Z)-3-hexen-1-ol] negatively affectedthe growth of SSB caterpillars (Fig. 5B). Thus, thereduction of herbivore resistance in as-pld rice plantscan at least partially be explained by a decrease ininduced TrypPI activity and GLV levels. Similar toour previous results found on as-lox rice mutants(Zhou et al., 2009), exogenous application of JA on as-pld plants also partially restored the induced TrypPIaccumulation, while GLVs did not (Fig. 4, C and D).This suggests that other herbivore-induced signals, inaddition to OsPLDa4- and -a5-derived JA, are in-

Figure 7. Mean percentage (+SE; n = 5) of BPH eggs per plant on pairs ofplants 48 h after exposure to different treatments: wild-type lanolintreatment (WT) versus L10-6 GLV treatment [containing (Z)-3-hexenaland (Z)-3-hexen-1-ol, each with 0, 125, 250, or 500 nmol; A]; wild-type lanolin treatment versus L10-6 GLV treatment [125 or 250 nmol of(Z)-3-hexenal or (Z)-3-hexen-1-ol; B]; wild-type lanolin treatmentversus wild-type GLV treatment [125 or 250 nmol of (Z)-3-hexenal]and wild-type sodium phosphate buffer (BUF) treatment versus L10-6JA treatment (C). Asterisks indicate significant differences between pairsof lines (* P , 0.05, ** P , 0.01, Student’s t test).

Qi et al.




volved in OsPLDa4- and -a5-dependent TrypPI ac-cumulation. Ethylene, for example, has been reportedto be involved in the production of herbivore-inducedrice TrypPIs (Wang et al., 2011). Given the obviousdecrease in ethylene levels in as-pld lines (Fig. 3),ethylene may be one of these additional signals.

Similar to the larval performance of SSB, femaleadults or nymphs of BPH, a homopteran phloemfeeder of rice, also showed a preference for settling,ovipositing, and feeding on as-pld plants rather thanon wild-type plants and survived better on the former(Fig. 6). Moreover, as-pld plants died more rapidly

Figure 8. Typical chromatograms ob-tained by head space collections fromnonmanipulated plants (control [Con])and SSB-infested plants (for 24 h) ofwild-type plants (WT) and as-pld linesL10-2 and L10-6. Numbers representchemicals and are the same as in TableI. [See online article for color versionof this figure.]

Table I. Volatile compounds emitted by wild-type plants and as-pld lines L10-2 and L10-6 by healthy plants and plants infested by SSB for 24 h

Data represent mean amounts (% of internal standard peak area) of five replications. Letters in the same row indicate significant differences amongtreatments (P , 0.05, Duncan’s multiple range test). Con, Control.

No. Chemical Wild-Type Con L10-2 Con L10-6 Con Wild-Type SSB L10-2 SSB L10-6 SSB

1 2-Heptanone 9.50 6 2.80 c 3.99 6 0.60 c 5.49 6 2.28 c 204.03 6 42.26 a 84.91 6 16.08 b 123.27 6 22.30 b

2 2-Heptanol 6.90 6 2.75 c 2.26 6 0.42 c 4.21 6 2.00 c 72.13 6 11.32 a 48.75 6 7.19 b 53.44 6 9.24 a,b

3 a-Thujene 0.53 6 0.09 b 0.62 6 0.06 b 0.80 6 0.20 b 3.70 6 0.58 a 3.01 6 0.17 a 3.27 6 0.40 a

4 a-Pinene 1.99 6 1.08 b 1.07 6 0.23 b 0.95 6 0.23 b 4.56 6 0.43 a 4.23 6 0.26 a 4.46 6 0.60 a

5 Myrcene 1.02 6 0.12 b 0.59 6 0.11 b 1.01 6 0.25 b 3.53 6 0.45 a 3.14 6 0.25 a 3.15 6 0.50 a

6 (+)-Limonene 11.89 6 0.78 c 9.24 6 1.47 b,c 14.52 6 3.35 c 58.82 6 7.22 a 52.77 6 3.58 a,b 55.33 6 9.43 a

7 (E)-Linalool oxide 1.71 6 0.31 c 1.00 6 0.22 c 0.98 6 0.33 c 36.55 6 8.04 a 10.37 6 2.18 b,c 17.84 6 5.80 b

8 Linalool 24.49 6 7.18 b 19.18 6 7.15 b 21.35 6 10.11 b 182.62 6 53.30 a 72.08 6 14.55 b 76.66 6 23.68 b

9 Methyl salicylate 5.20 6 1.32 b 2.58 6 0.84 b 3.12 6 1.57 b 24.25 6 5.75 a 4.45 6 0.40 b 5.89 6 2.55 b

10 Unknown 1 4.06 6 1.92 b 1.66 6 0.87 b 1.24 6 0.21 b 10.60 6 1.05 a 1.73 6 0.10 b 3.16 6 0.79 b

11 Unknown 2 1.99 6 0.45 b 1.02 6 0.43 b 1.86 6 1.07 b 13.57 6 4.49 a 4.03 6 0.68 b 5.44 6 1.26 b

12 a-Copaene 0.46 6 0.16 b 0.40 6 0.08 b 0.54 6 0.20 b 5.54 6 0.82 a 2.98 6 0.34 b 3.79 6 1.67 b

13 Unknown 3 0.73 6 0.30 b 0.24 6 0.13 b 0.20 6 0.03 b 1.50 6 0.23 a 0.22 6 0.04 b 0.24 6 0.04 b

14 Sesquithujene 1.83 6 0.17 a 1.14 6 0.20 a 1.33 6 0.27 a 3.74 6 0.69 a 2.64 6 0.22 a 2.24 6 0.39 a

15 a-Cedrene 4.53 6 1.86 a 1.79 6 0.42 a 1.39 6 0.22 a 3.74 6 0.33 a 1.46 6 0.18 a 0.83 6 0.16 a

16 (E)-b-Caryophyllene 4.50 6 0.17 b 2.97 6 1.09 b 2.82 6 0.73 b 9.93 6 1.35 a 5.43 6 0.80 b 3.58 6 0.84 b

17 (E)-a-Bergamotene 1.43 6 0.58 b 0.86 6 0.30 b 1.11 6 0.50 b 10.44 6 2.79 a 5.18 6 0.98 b 5.24 6 1.23 b

18 Sesquisabinene A 3.23 6 0.17 b,c 1.75 6 0.60 c 2.21 6 0.62 c 13.03 6 2.94 a 6.91 6 0.96 b 6.68 6 1.36 b

19 (E)-b-Farnesene 6.83 6 1.29 b,c 2.55 6 0.83 c 3.85 6 0.91 b,c 18.85 6 3.17 a 7.80 6 0.23 b 6.41 6 1.51 b,c

20 a-Curcumene 1.40 6 0.28 b 1.70 6 0.57 b 1.18 6 0.37 b 14.38 6 3.83 a 5.53 6 0.79 b 6.05 6 1.45 b

21 Zingiberene 13.39 6 1.37 b 6.50 6 1.66 b 10.10 6 2.10 b 39.33 6 10.80 a 18.82 6 0.77 b 17.63 6 3.44 b

22 b-Bisabolene 2.10 6 0.59 b 1.36 6 0.44 b 1.71 6 0.72 b 12.76 6 4.58 a 7.33 6 1.28 a,b 7.02 6 1.40 a,b

23 b-Sesquiphellandrene 2.86 6 0.95 b 1.69 6 0.69 b 2.24 6 0.98 b 19.92 6 6.76 a 11.25 6 2.02 a,b 11.09 6 2.17 a,b

24 (E)-g-Bisabolene 1.49 6 0.31 b 0.80 6 0.12 b 1.53 6 0.44 b 7.99 6 3.26 a 5.09 6 0.55 a,b 3.77 6 0.70 b





than wild-type plants when infested with an equalnumber of BPH female adults (Fig. 6G). This findingcontrasts with our previous results showing that as-loxplants, which had lower elicited JA levels than wild-type plants, were more resistant to BPH (Zhou et al.,2009). This difference may be related to the levels ofH2O2 and GLVs. Compared with wild-type plants, as-lox plants had higher levels of BPH-induced H2O2 andequal GLV levels (Zhou et al., 2009), whereas as-pldplants had equal H2O2 levels (Supplemental Fig. S7)and lower elicited GLV levels (Fig. 3). The GLV (Z)-3-hexen-1-ol repelled BPH (Fig. 7B; Supplemental Fig. S8,D and E), while JA had no obvious effect on BPHpreference (Fig. 7C; Supplemental Fig. S8J). Therefore,the increased resistance to BPH in as-lox plants mightbe related to its higher BPH-inducedH2O2 levels, which,together with elicited SA, induce a hypersensitiveresponse-like cell death that inhibits BPH feeding(Zhou et al., 2009), whereas the reduced resistance inas-pld lines may be the result of reduced (Z)-3-hexen-1-ollevels.

Apart from their direct effects, herbivore-inducedplant volatiles (HIPVs) also attract natural enemies ofthe herbivores, thereby indirectly protecting plants(Kessler et al., 2004; Frost et al., 2008; Dicke, 2009). Forthe production of HIPVs, JA signaling plays a centralrole (Kessler et al., 2004; Dicke, 2009). Here, we alsofound that as-pld plants, which had reduced elicited JAand ethylene levels compared with wild-type plants(Fig. 3, A and C), released lower levels of inducedvolatiles than wild-type plants did (Fig. 8; Table I),followed by a reduced attractiveness to the larvalparasitoid of SSB, A. chilonis (Fig. 5C). Previous exper-iments have shown that exogenous application of JAon rice plants induced volatile emission (Lou et al.,2005). Moreover, volatiles emitted from SSB-infestedplants were attractive to the parasitoid A. chilonis(Chen et al., 2002). Together with these findings, ourresults demonstrate that OsPLDa4 and -a5 are in-volved in the SSB-induced, volatile-mediated rice in-direct defense via increases of the biosynthesis of JA.

In summary, OsPLDa4 and -a5 can regulate the riceoxylipin and ethylene signaling cascade, including JA,GLVs, and ethylene, possibly by influencing the levelsof LeA, the activity of LOX and ACS, and MAPKsignaling, which, taken together, results in an effectiveherbivore-induced direct and indirect defense re-sponse. Our study provides a compelling example ofhow genes that act at the very beginning of the wound-and herbivore-induced oxylipin and ethylene path-ways can influence plant-insect interactions up to thethird trophic level.

MATERIALS AND METHODS

Plant Growth

The rice (Oryza sativa) genotypes used in this study were Xiushui 11 wild

type and as-pld transgenic lines (see below). Pregerminated seeds were

cultured in plastic bottles (diameter, 8 cm; height, 10 cm) in a greenhouse

(28�C 6 2�C, 14 h of light and 10 h of dark). Ten-day-old seedlings were

transferred to 50-L communal hydroponic boxes with a rice nutrient solution

(Yoshida et al., 1976). After 30 to 35 d, seedlings were transferred to individual

500-mL hydroponic plastic pots, each pot with one or two plants (for two

plants, one was a wild-type plant and the other was an as-pld transgenic line

plant). Plants were used for experiments 4 to 5 d after transplanting.

Insects

Colonies of SSB (Chilo suppressalis) and BPH (Nilaparvata lugens) were

maintained on Shanyou 63 (a variety susceptible to SSB and BPH) rice

seedlings using the same method as described by Zhou et al. (2009). Apanteles

chilonis (Munakata) colonies were obtained from rice fields in Hangzhou,

China, and maintained on SSB larvae.

Generation and Characterization of as-pldTransgenic Lines

A 1,060-bp portion (Supplemental Fig. S1) of the OsPLDa4 cDNA present

on plasmid pPLDa4 (see below) was PCR amplified using the primers

5#-CAGTCGCTCGGCATCAAG-3# and 5#-TTCCCAAGCAGAAGAAGGTG-3#,which have 91% sequence similarity with OsPLDa5, and then was cloned into

pCAMBIA1301, yielding an antisense transformation vector (Supplemental

Fig. S4). The vector was inserted into the rice variety Xiushui 11 using

Agrobacterium tumefaciens-mediated transformation. The procedure of rice

transformation, screening of the homozygous T2 plants, and identification of

the number of insertions followed the same method as described by Zhou

et al. (2009). For most experiments, two T2 homozygous lines, L10-2 and L10-6,

each harboring a single insertion (Supplemental Fig. S5), and wild-type plants

were used.

Plant Treatments

Pots with one plant were used for this experiment. For SSB treatment,

plants were individually infested using a third-instar larva of SSB that had

been starved for 2 h. Control plants were left herbivore free. For BPH

treatment, plants were individually infested with 15 gravid BPH females that

were confined in a glass cylinder (diameter, 4 cm; height, 8 cm; with 48 small

holes [diameter, 0.8 mm]). One empty cylinder was attached to control plants

(noninfested). For mechanical wounding, plants were individually damaged

using a needle on the lower part of rice stems (about 2 cm long) with 200 holes.

Control plants were not pierced. The method for JA and SA treatment was the

same as described by Zhou et al. (2009). Plants were individually sprayedwith

2 mL of JA (100 mg mL21) or SA (70 mg mL21) in 50 mM sodium phosphate

buffer. Control plants were sprayed with 2 mL of buffer. For GLV treatment,

plants from each genotype were individually treated with 125 to 500 nmol of

(Z)-3-hexenal or (Z)-3-hexen-1-ol in lanolin paste (see details for each exper-

iment) on stems. Control plants received the same volume of pure lanolin

paste.

Isolation of the Full-Length cDNA of OsPLDa4 and -a5

Full-length cDNA of a SSB-induced OsPLDa4 and -a5 were obtained by

RT-PCR from total RNA isolated from wild-type plants infested by SSB larvae

for 24 h. The primers PLDa4-F (5#-CTGCTTCTTCTTCCCGTTCTT-3#), PLDa4-R

(5#-GCAGGTGCATACTCATCATTAC-3#), PLDa5-F (5#-ATTGTTTTGCGAG-

CTTCTTCC-3#), and PLDa5-R (5#-CGATGAACATAATTCATATTAGA-3#)were designed based on the sequences of the rice PLD genes (accession nos.

AK100278 and AK119861 for OsPLDa4 and -a5; Yamaguchi et al., 2009), which

have high homology with the partial sequences of the two OsPLD genes that

were cloned by suppression subtractive hybridization (G. Zhou and Y. Lou,

unpublished data). The PCR products were cloned into pMD19-T vector

(Takara; http://www.takara.com.cn/) and sequenced.

qRT-PCR

For qRT-PCR analysis, five independent biological samples were used.

Total RNAwas isolated using the SV Total RNA Isolation System (Promega).

Fifty nanograms of each total RNA sample was reverse transcribed using the

PrimeScript RT-PCR Kit (TaKaRa). qRT-PCR was performed on an ABI PRISM

7500 sequence detection system (Applied Biosystems) with OsACT RNA for

Qi et al.




normalization. The primers, TaqMan probe sequences used for TaqMan qRT-

PCR (Premix Ex Taq Kit; TaKaRa), and primer sequences for SYBR Green-

based qRT-PCR (SYBR Premix ExTaq; TaKaRa) are shown in Supplemental

Table S1. The relative expression levels of the target genes were determined

using standard curves (Livak, 1997).

JA, SA, Ethlyene, and GLVAnalysis

Plants (one plant per pot) were randomly assigned to SSB or control

treatment. Two or three as-pld lines (L10-2, L10-6, and L10-22) and one wild-

type line were used. The stems were harvested at 0, 1.5, and 3 h after SSB

treatment, and JA and SA levels were analyzed by gas chromatography-mass

spectrometry using labeled internal standards as described by Lou and

Baldwin (2003). Plants were individually covered with a sealed glass cylinder

(diameter, 4 cm; height, 50 cm), and ethylene production was determined

using the same method as described by Lu et al. (2006). Each treatment at each

time interval was replicated five to six times.

GLV emissions [(Z)-3-hexenal and (Z)-3-hexen-1-ol] were analyzed with a

portable gas analyzer (zNose 4200; Electronic Sensor Technology; http://

www.estcal.com) using the same method as described by Zhou et al. (2009).

Five (controls) and 13 (wounding) replicates were carried out for each

genotype (L10-2, L10-6, and one wild-type line).

Quantification of H2O2

Wild-type plants and plants of as-pld lines L10-2 and L10-6 were randomly

assigned to BPH or control treatment. Leaf sheaths were harvested at 0, 3, 8,

and 24 h after treatment. Each treatment at each time interval was replicated

five times. H2O2 concentrations were determined as described by Lou and

Baldwin (2006).

LeA Analysis

We compared LeA levels in two as-pld lines and one wild-type line at 0, 15,

30, 60, and 120 min after SSB treatment. For each treatment and each time

interval, stems (0.15 g) of five plants were sampled. LeAwas extracted for gas

chromatography-mass spectrometry analysis, which was similar to the

method described by Qu et al. (2006) with a small modification. Samples

were individually added with 10 mL of ethyl decanoate (2,000 ppm) as internal

standard before LeA extraction, and the extracts were derivatized with

diazomethane. Quantification of LeA in samples was achieved according to

a standard curve that was obtained by authentic standard LeA.

TrypPI Analysis

Plants (one plant per pot) from each line (L10-2, L10-6, and the wild-type

line) were randomly assigned to nine groups (Fig. 4). For JA + SSB and buffer +

SSB treatment, the plants were treated with either JA or buffer for 1 d,

followed by infestation of SSB third-instar larvae (one larva per plant); for (Z)-

3-hexenal + SSB, (Z)-3-hexen-1-ol + SSB, and lanolin + SSB treatment, the

plants were treated with 250 nmol of (Z)-3-hexenal, (Z)-3-hexen-1-ol, or

lanolin, immediately followed by infestation of SSB third-instar larvae (one

larva per plant). Stems (0.12–0.15 g per sample) were harvested 3 d after the

start of herbivore infestation. The TrypPI concentrations were measured using

a radial diffusion assay as described by van Dam et al. (2001). Each treatment

at each time interval was replicated five times.

Collection, Isolation, and Identification of Rice Volatiles

The collection, isolation, and identification of rice volatiles used the same

method as described by Lou et al. (2005). Volatiles emitted from individual

plants (one plant per pot) of each line (L10-2, L10-6, and the wild-type line)

that were infested with SSB for 24 h or nonmanipulated were collected.

Collections were replicated five times for each treatment. The compounds

were expressed as percentage of peak areas relative to the internal standard

per 8 h of trapping for one plant.

Olfactometer Bioassays

Responses of A. chilonis (Munakata) females to rice volatiles were mea-

sured in a Y-tube olfactometer using the same method as described by Lou

et al. (2005). The behavioral response of the parasitoid exposed to the

following pairs of odor sources was observed: SSB-infested plants of each

as-pld line (L10-2 and L10-6) versus SSB-infested wild-type plants (infestation

for 24 h); control plants of each line versus control wild-type plants. For each

treatment, 10 plants were used, and the odor sources were replaced by a new

set of 10 plants after testing eight wasps. For each odor source combination, a

total of 48 females were tested.

Herbivory Experiments

Seven-day-old larvae of SSB, which had been weighed and starved for 2 h,

were placed individually on each plant of the two as-pld lines (L10-2 and L10-

6) and the wild-type line. To test the effect of GLVs on the larval performance

of SSB, the larvae were placed individually on each wild-type plant that was

treated with either GLVs [10 mL of lanolin containing (Z)-3-hexenal and (Z)-3-

hexen-1-ol, at 250 nmol each] or lanolin only (10 mL of lanolin). Twenty-four to

30 replicate plants from each line and treatment were used. Larval mass (to an

accuracy of 0.1 mg) was measured 7 d after the start of the experiment, and the

increased percentage of larval mass on each line or treatment was calculated.

To determine the colonization and oviposition preference of BPH, pots

with two plants (an as-pld line plant versus a wild-type plant) were confined

with glass cylinders into which 15 gravid adult BPH females or nymphs were

introduced. In complementation experiments, transgenic line L10-6 and wild-

type plants were used; the colonization and oviposition preferences of BPH

female adults were determined for 11 pairs of plants (Fig. 7). The numbers of

BPH at different times and BPH eggs at 48 h on each plant were counted

followed the method described by Zhou et al. (2009). The experiment was

repeated five to six times.

The survival rates of BPH nymphs on wild-type and as-pld plants were

recorded 6 d after the introduction of the herbivore using the method

described by Zhou et al. (2009). The experiment was repeated eight times.

The differences in plant tolerance to herbivore attack between wild-type

and as-pld plants were determined following the method described by Zhou

et al. (2009).

Data Analysis

Differences in SSB-induced JA, SA, ethylene, LeA and volatiles, TrypPIs,

and GLVs as well as expression levels of OsHI-LOX, OsACS2, and OsHPL3

were analyzed by one-way ANOVA. If the ANOVAwas significant (P, 0.05),

Duncan’s multiple range test was used to detect significant differences

between groups. Differences in the attractiveness of HIPVs to the parasitoid

between lines were tested by x2. Differences in experiments using two

treatments were determined by Student’s t test. Data were analyzed with

Statistica (SAS Institute).

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers OsPLDa1, AK065102; OsPLDa2, AK240654;

OsPLDa3, AK072121; OsPLDa4, AK100278; OsPLDa5, AK119861; OsPLDh1,

AC099323; OsPLDh2, AP005388; OsHI-LOX, FJ607153; OsHPL3, AK105694;

OsACS2, AK064250; OsMPK3, AF479883; OsMPK6, AJ535841; and OsACT,

AB047313.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. cDNA sequence alignment of the two cloned

PLD genes (OsPLD1 andOsPLD2), AK100278 (OsPLDa4) and AK119861

(OsPLDa5).

Supplemental Figure S2. Alignment of deduced amino acid sequences of

the two cloned PLD genes, OsPLD1 and OsPLD2.

Supplemental Figure S3. Mean expression levels (relative to expression

levels of OsACT; +SE; n = 5) of OsPLDa4 and -a5 in rice stems after

different treatments.

Supplemental Figure S4. Rice transformation vector pCAMBIA-PLD (13.7

kb) with hgh and gus as plant-selectable marker genes.

Supplemental Figure S5. DNA gel-blot analysis and growth phenotypes

of as-pld lines and wild-type plants.

Supplemental Figure S6. Mean expression levels (relative to expression

levels ofOsACT; +SE; n = 5) ofOsPLDa1,OsPLDa2,OsPLDa3,OsPLDh1,





OsPLDh2, andOsMPK6 in stems of wild-type plants and two as-pld lines

at different times after SSB infestation.

Supplemental Figure S7. Mean H2O2 concentrations (+SE; n = 5) in as-pld

lines L10-2 and L10-6 and wild-type plants at various times after plants

were individually infested with 15 BPH female adults or not infested.

Supplemental Figure S8. Mean number of BPH female adults per plant

(+SE; n = 5) on pairs of plants with different treatments, 48 h after five

replicated plant pairs were exposed to 15 female adults.

Supplemental Table S1. Primers and probes used for qRT-PCR of target

genes.

ACKNOWLEDGMENTS

We thank Tobias Kollner for his invaluable assistance with the identifica-

tion of volatiles.

Received July 20, 2011; accepted October 6, 2011; published October 7, 2011.

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