OsNPR1 negatively regulates herbivore-induced JA and ethylene signaling and plant resistance to a chewing herbivore in rice

Physiologia Plantarum 2012 Copyright © Physiologia Plantarum 2012, ISSN 0031-9317

OsNPR1 negatively regulates herbivore-induced JA andethylene signaling and plant resistance to a chewingherbivore in riceRan Li, Sumera Afsheen, Zhaojun Xin, Xiu Han and Yonggen Lou

State Key Laboratory of Rice Biology, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China

Correspondence*Corresponding author,e-mail: [email protected]

Received 20 April 2012;revised 26 May 2012

doi:10.1111/j.1399-3054.2012.01666.x

NPR1 (a non-expressor of pathogenesis-related genes1) has been reportedto play an important role in plant defense by regulating signaling pathways.However, little to nothing is known about its function in herbivore-induceddefense in monocot plants. Here, using suppressive substrate hybridization,we identified a NPR1 gene from rice, OsNPR1, and found that its expressionlevels were upregulated in response to infestation by the rice striped stem borer(SSB) Chilo suppressalis and rice leaf folder (LF) Cnaphalocrocis medinalis,and to mechanical wounding and treatment with jasmonic acid (JA) andsalicylic acid (SA). Moreover, mechanical wounding induced the expressionof OsNPR1 quickly, whereas herbivore infestation induced the gene moreslowly. The antisense expression of OsNPR1 (as-npr1), which reduced theexpression of the gene by 50%, increased elicited levels of JA and ethylene(ET) as well as of expression of a lipoxygenase gene OsHI-LOX and anACC synthase gene OsACS2. The enhanced JA and ET signaling in as-npr1plants increased the levels of herbivore-induced trypsin proteinase inhibitors(TrypPIs) and volatiles, and reduced the performance of SSB. Our resultssuggest that OsNPR1 is an early responding gene in herbivore-induceddefense and that plants can use it to activate a specific and appropriatedefense response against invaders by modulating signaling pathways.

Introduction

In nature, plants suffer from many biotic stresses, suchas pathogens and herbivores. To protect themselvesfrom damage by invaders, plants have evolved aseries of defense mechanisms, both constitutive andinducible (Pieterse and van Loon 2004). It has beenwell documented that jasmonic acid (JA), salicylic acid(SA) and ethylene (ET) signaling pathways as well asthe interactions among these compounds play a centralrole in shaping induced plant defenses (Koornneef and

Abbreviations – ACC, 1-aminocyclopropane-1-carboxylate; ACS, 1-aminocyclopropane-1-carboxylate synthase; ET,ethylene; HIPV, herbivore-induced volatile; IS, internal standard; JA, jasmonic acid; LF, leaf folder; NPR1, non-expressor of pathogenesis-related genes1; PCR, polymerase chain reaction; QRT-PCR, quantitative real-time polymerasechain reaction; RT-PCR, reverse transcription polymerase chain reaction; SA, salicylic acid; SSB, striped stem borer; SSH,suppression subtractive hybridization; TrypPIs, trypsin proteinase inhibitors; WT, wild type.

Pieterse 2008). So far, several components involved incrosstalk between SA and JA, such as the non-expressorof pathogenesis-related genes1 (NPR1), WRKY70 andMPK4, have been identified, and these componentshave been reported to fine-tune-specific plant defenseresponses by mediating different pathways (Petersen etal. 2000, Spoel et al. 2003, Li et al. 2004).

NPR1 was first identified from an Arabidopsis mutantimpaired in its systemic acquired resistance (SAR) (Cao etal. 1994, 1997). It has long been recognized that NPR1is a major regulator of pathogen-induced SA-mediated

Physiol. Plant. 2012

SAR and is involved in plant defense against a broadspectrum of pathogens (Cao et al. 1998, Fitzgerald et al.2004, Chern et al. 2005, Wally et al. 2009, Parkhi et al.2010). When plants are infected by a pathogen, whichcauses an accumulation of SA and changes in cellularredox, NPR1 changes from inactive oligomers (present incytoplasm) to active monomers; these then translocate tothe nucleus where they interact with TGA transcriptionfactors and regulate the expression of PR genes (Despreset al. 2000, Fan and Dong 2002, Mou et al. 2003). Dur-ing this process, proteasome-mediated turnover of NPR1plays an important role in modulating transcription of itstarget genes (Spoel et al. 2009). Moreover, some com-ponents, such as AtNUDX6, an ADP-ribose (Rib)/NADHpyrophosphohydrolase and nitric oxide in Arabidopsisthaliana were reported to be important regulator of theNPR1-dependent defense pathway (Isikawa et al. 2010,Lindermayr et al. 2010). Recently, NPR1 has also beenfound to be joined to various defense-related signalingpathways. In A. thaliana, for example, cytosol-localizedNPR1 is required for the SA-mediated suppressionof JA-dependent defenses (Spoel et al. 2003) and ETmediates this interaction by modulating the role ofNPR1 (Leon-Reyes et al. 2009); when attacked by apathogen, NPR1 negatively regulates SA biosynthesis(Shah 2003). In Nicotiana attenuata, NPR1 was found tosuppress SA biosynthesis during herbivore infestation;suppressed SA biosynthesis is known to enhance JAproduction (Rayapuram and Baldwin 2007). Duringinduced systemic resistance in A. thaliana, NPR1regulates cross-communication between JA and ETsignaling, independently of SA (Pieterse and van Loon2004). These studies suggest that NPR1 plays diverseroles in modulating defense-related signaling cascades.

Consistent with its different effects on signalingpathways in various plant species, NPR1 plays differentroles in plant-herbivore resistance according to thespecies. In Arabidopsis, for instance, as NPR1 negativelyregulates JA levels, the npr1 mutant enhances direct(Cui et al. 2002, Stotz et al. 2002, Mewis et al. 2006)and indirect (Girling et al. 2008) plant resistance toherbivores. In N. attenuata, as NPR1 positively mediatesJA biosynthesis by inhibiting SA biosynthesis, silencingNPR1 decreases plant resistance to the generalistlepidopteran herbivore Spodoptera exigua in natureand in the glasshouse, and also impairs the ability ofplants to attract the natural enemies of the herbivoresin nature (Rayapuram and Baldwin, 2007). Similarly,over-expressing Arabidopsis NPR1 in tobacco Nico-tiana tabacum enhances plant resistance to the earlyinstars of Spodoptera litura (Meur et al. 2008). Theseresults show that NPR1 can positively or negativelymodulate herbivore-induced direct and indirect defense

responses. However, up to now, these studies havemainly focused on dicot plants, such as Arabidopsis. Weinvestigate the function of NPR1 in monocot plants inplant defense, especially in herbivore-induced defense,a topic that has remained unexplored until now.

Rice, one of the most important food crops in theworld, suffers heavily from insect pests (Cheng andHe 1996). Previous studies with rice have shown thatherbivore attack induces a variety of plant hormonesincluding JA, SA and ET, which subsequently regulatedefensive responses, including the release of herbivore-induced volatiles (HIPVs) and the accumulation oftrypsin proteinase inhibitors (TrypPIs) (Lou et al. 2005a,2005b, 2006, Lu et al. 2006, Zhou et al. 2009, Lu et al.2011). Given the important role of NPR1 in mediatingcross-communication between signaling pathways, asstated above, it is not surprising that OsNPR1 (alsocalled NH1) may also play a central role in herbivore-induced rice defense responses. It has been reported thattranscript levels of OsNPR1 are upregulated followinginfection by the bacterial blight Xanthomonas oryzaepv. oryzae (Xoo), the rice fungal blast Magnaporthegrisea and treatment with signal molecules, such asbenzothiadiazole, methyl jasmonate and ET (Yuan etal. 2007). OsNPR1 positively regulates expression ofPR genes (Shimono et al. 2007, Feng et al. 2011) andrice resistance to bacterial blight disease (Yuan et al.2007) and fungal blast disease (Shimono et al. 2007,Sugano et al. 2010, Feng et al. 2011) but negativelymodulates resistance to the white-backed planthopperSogatella furciferia (Yuan et al. 2007). Recently, Suganoet al. (2010) found that OsNPR1 may play a role inthe reallocation of energy and resources during defenseresponses. However, how OsNPR1 mediates defense-related signaling pathways and thus regulates the abilityof rice to resist herbivores remains largely unknown.

To explore these issues, we isolated a rice NPR1 gene,OsNPR1, which is induced by herbivore infestation.Using a reverse genetics approach, we obtained ricelines with antisense expression of OsNPR1 (as-npr1). Wedetermined the role of OsNPR1 in herbivore-induceddefense by measuring the expression of JA and ETbiosynthesis-related genes, and the levels of phytohor-mones and defense-related chemicals as well as theresistance to a chewing herbivore, the striped stem borer(SSB), Chilo suppressalis. Our results provide evidencethat OsNPR1 is involved in rice herbivore defense.

Materials and methods

Plant growth and insects

The rice genotypes used in this study were the japonicarice variety Xiushui 11 wild type (WT, untransformed)


and two as-npr1 transgenic lines (see below) in the samebackground. Pre-germinated seeds were cultured inplastic bottles (8 cm diameter, 10 cm height) in a green-house (28 ± 2◦C, 14 L: 10D). Ten-day-old seedlingswere transferred to 50-l communal hydroponic boxeswith a rice nutrient solution (Yoshida et al. 1976). After30–35 days, seedlings were transferred to individual500-ml hydroponic plastic pots. Plants were used forexperiments 4–5 days after transplanting. Colonies ofSSB and leaf folder (LF) were originally obtained fromrice fields in Hangzhou, China, and maintained onXiushui 11 rice seedlings using the same method asdescribed in Zhou et al. (2009).

Generation and characterization of as-npr1transgenic lines

A 623 bp portion (see Fig. S1) of the OsNPR1cDNA present on plasmid pNPR1 (see below) waspolymerase chain reaction (PCR)-amplified using theprimers 5′-TAGGATCCGGGTTACCGAAGAAGGA-3′

and 5′-GGTCTAGAGGCAACTATTTGGGAGCA-3′, andthen was digested by BamHI and XbaI, and cloned intopCAMBIA1301, yielding an antisense transformationvector (see Fig. S2). The vector was inserted into the ricevariety Xiushui 11 using Agrobacterium tumefaciens-mediated transformation. The procedure of transformingrice, screening the homozygous T2 plants and iden-tifying the number of insertions followed the methoddescribed in Zhou et al. (2009). For most experiments,two T2 homozygous lines, N6 and N7, each harboring asingle insertion (see Fig. S3A), and WT plants were used.

Plant treatments

Mechanical wounding

Plants (one per pot) were individually damaged using aneedle to pierce the lower part of rice stems (about 2 cmlong), with 200 holes (W). Control plants (Con) were notpierced.

SSB treatment

Plants (one per pot) were individually infested using athird-instar larva of SSB that had been starved for 2 h.Control plants (Con) were left herbivore-free.

LF treatment

Plants (one per pot) were individually infested usinga third-instar larva of LF that had been starved for 2h and then placed on leaves at node 3 (the youngestfully expanded leaf was defined as leaf node 1). Controlplants (Con) were left herbivore-free.

JA and SA treatment

The method for JA and SA treatment was the same asdescribed in Zhou et al. (2009). Plants were individuallysprayed with 2 ml of JA (100 μg ml−1) or SA (70 μg ml−1)in 50 mM sodium phosphate buffer. Control plants weresprayed with 2 ml of buffer (Buf).

Isolation of the full-length cDNA of OsNPR1

Full-length cDNA of an SSB-induced OsNPR1 wereobtained by Reverse transcription PCR (RT-PCR) fromtotal RNA isolated from WT plants infested by an SSBlarva for 24 h. The primers NPR1-F (5′-AATGGAGCCGCCGACCAGCCA-3′) and NPR1-R (5′-GGCAACCACAGGTGAGAGCT-3′) were designed based on the sequenceof the rice NPR1 gene (Genebank number: DQ450947),which have high homology with the partial sequencesof the NPR1-like gene that were cloned by suppressionsubtractive hybridization (SSH). The PCR products werecloned into pMD19-T vector (TaKaRa, Dalian, China)(pNPR1) and sequenced.

QRT-PCR analysis

For quantitative RT-PCR (QRT-PCR) analysis, three tofour independent biological samples were used. TotalRNA was isolated using the SV Total RNA IsolationSystem (Promega, Madison, WI). One microgram ofeach total RNA sample was reverse-transcribed using thePrimeScript™ RT-PCR Kit (TaKaRa, Dalian, China). qRT-PCR was performed on a CFX96TM Real-Time system(Bio-RAD, California, USA) using Premix Ex TaqTM

Kit (TaKaRa, Dalian,China). The primers and probesequences used for mRNA detection of target genesby qRT-PCR are shown in Table S1. A rice actin geneOsActin (TIGR ID: Os03g50885) was used as an internalstandard (IS) to normalize cDNA concentrations.

SSB performance measurement

Second-instar larvae of SSB, which had been weighedand starved for 2 h, were placed individually on eachplant of the two as-npr1 lines (N6 and N7) and the WTline. Thirty replicate plants from each line were used.Larval mass (to an accuracy of 0.1 mg) was measured 7days after the start of the experiment and the increasedpercentages of larval mass on each line were calculated.

TrypPI analysis

Plants (one plant per pot) from each line (N6, N7 andWT line) were randomly assigned to control (Con), SSBand LF groups. Stems (SSB infestation) or leaves (LF


infestation) (0.2–0.3 g per sample) were harvested 3days after the start of the treatment. The TrypPI activitywas measured using a radial diffusion assay as describedin van Dam et al. (2001). Each treatment was replicatedfive times.

SA, JA and ET analysis

Plants (one plant per pot) were randomly assignedto SSB and control treatment. Two as-npr1 lines (N6and N7) and one WT line were used. The stems wereharvested at 0 and 3 h after SSB treatment, and JA andSA levels were analyzed by gas chromatography-massspectrometry using labeled ISs as described in Louand Baldwin (2003). Three plants were covered witha sealed glass cylinder (4 cm diameter, 50 cm height)and ET production was determined at 12 and 24 h afterthe start of the experiment using the same method asdescribed in Lu et al. (2006). Each treatment at eachtime interval was replicated five to six times.

Collection, isolation and identificationof rice volatiles

The collection, isolation and identification of rice volatileused the same method as described in Lou et al. (2005a).Volatiles emitted from individual plants (one plant perpot) of each line (N6, N7 and WT lines) that were infestedwith SSB for 24 h or from non-manipulated plantswere collected. Collections were replicated five timesfor each treatment. The compounds were expressed aspercentages of peak areas relative to the IS (diethylsebacate) per 8 h of trapping using one plant.

Data analysis

Differences in the herbivore-elicited activity of TrypPIsand volatiles were analyzed by one-way ANOVA; if theANOVA analysis was significant (P < 0.05), Duncan’smultiple range tests were used to detect significantdifference between groups. Differences in experimentsusing two treatments were determined by Student’s t-tests. Data were analyzed with STATISTICA (Statistica; SASInstitute Inc., Cary, NC).

Results

Isolation and characterization of OsNPR1

Using SSH, we screened rice plants for herbivore-induced transcripts. Using this technique, we identifieda clone that showed similarity to OsNPR1. The full-length cDNA of the cloned SSB-induced OsNPR1-likegene, including an open reading frame of 1749 bp, was

then obtained by RT-PCR (see Table S1). Nucleotideblast analysis showed that the sequence was completelyidentical (100 %) to that of the previously identifiedOsNPR1 (Chern et al. 2005), suggesting that the isolatedgene is OsNPR1.

QRT-PCR analysis revealed low constitutive expres-sion of OsNPR1, while mechanical wounding and SAtreatment resulted in a rapid increase (<1 h) in transcriptlevels (Fig. 1A, B). SSB or LF caterpillar infestation and JAtreatment also increased the expression of OsNPR1, buttheir elicitation was slow (Fig. 1A, C, D). These resultsshow that OsNPR1 might be involved in herbivore-induced defense responses in rice.

Antisense expression of OsNPR1 increases elicitedlevels of JA and ET but not SA

To determine the role of OsNPR1 in herbivore-induceddefense responses, we constructed pCAMBIA-1301transformation vectors carrying an antisense fragment(approximately 0.6 kb coding regions) of OsNPR1 (seeFig. S1), and generated transgenic rice plants usingA. tumefaciens-mediated transformation. By GUS stain-ing and hygromycin resistance selection, we obtainedfour T2 homozygous lines, all of which contained a singleT-DNA insertion (see Fig. S3A). Transcriptional analysisshowed that SSB-induced transcript levels of OsNPR1in as-npr1 lines N6 and N7 were about half of those inWT plants at 8 h (the time when the expression levelof OsNPR1 is the highest when plants were infested bySSB; Fig. 1C) after SSB infestation (Fig. 2A). In rice, geneswhose nucleotide sequences have the highest similarityto OsNPR1 are OsNPR2 and OsNPR3; however, both ofthem only share 51.48 and 53.06% sequence similaritywith OsNPR1 (see Table S1). Moreover, there are nomore than 10 consecutive nucleotides in the sequenceused for antisense transformation that are identical tothose in sequences of OsNPR2 or OsNPR3 (see TableS1). This suggests that the specificity of the antisensesequence is high, and the antisense construct shouldnot co-silence the transcript accumulation of any otherrice genes. There was no obvious difference in growthphenotype between WT plants and as-npr1 lines duringtheir lifetime (see Fig. S3B–D). For most of the followingexperiments, we compared two as-npr1 lines (N6 andN7) with untransformed WT plants.

Plant hormone signaling pathways play an importantrole in herbivore-induced defense responses (Wu andBaldwin 2009). Therefore, we measured the SSB-elicitedlevels of JA, SA and ET in WT plants and as-npr1lines. The results show that basal JA levels were similarbetween as-npr1 lines and WT plants, whereas JA levelsin as-npr1 lines were significantly increased compared


A B

C D

Fig. 1. Expression of OsNPR1 in rice stems or leaves after different treatments. (A, B, D) Mean transcript levels (+SE, n = 3–4) of OsNPR1 in ricestems that were treated with either jasmonic acid (JA), salicylic acid (SA), or a buffer (50 mM phosphate buffer, pH = 8.0) (Buf) (A), mechanicallywounded (B) or by the rice striped stem borer (SSB) (D). (C) Mean transcript levels (+SE, n = 4) of OsNPR1 in rice leaves that were infested by therice leaf folder (LF). Con, non-manipulation. Transcript levels were analyzed by QRT-PCR. Asterisks indicate significant differences in transcript levelsbetween treatments and controls (*P< 0.05; **P< 0.01; Student’s t-test).

to in WT plants upon SSB attack; the levels of JA inas-npr1 lines, N6 and N7 were 128.9 and 128.6% ofthose in WT plants 3 h after SSB infestation (Fig. 2C).Similarly, the levels of ET accumulation in as-npr1 lineswere higher than those in WT plants 12–24 h after SSBinfestation; at 24 h, the levels of ET in as-npr1 lines N6and N7 were 1.37- and 1.33-fold higher than those inWT plants (Fig. 2E). However, there was no difference inSA levels between as-npr1 lines and WT plants (Fig. 2B).In accordance with the results of JA and ET, silencingOsNPR1 enhanced the mRNA levels of OsHI-LOX (start-ing <1 h after infestation, Fig. 2D), a 13-lipoxygenasegene that is involved in herbivore-induced JA biosynthe-sis in rice (Zhou et al. 2009) and OsACS2 (starting <1 hafter infestation, Fig. 2F), an ACC synthase gene that isinvolved in herbivore-induced rice ET production (Lu etal. 2011). These results suggest that OsNPR1 modulatesherbivore-elicited JA and ET biosynthesis, but not SA.

Antisense expression of OsNPR1 enhancesherbivore-induced levels of TrypPIs and volatiles

TrypPIs and HIPVs are important direct and indirectdefense chemicals in rice against herbivores, and theirproduction is regulated by JA, SA and ET signalingpathways (Zhou et al. 2009, Lu et al. 2011, Qi et al.

2011, Wang et al. 2011). Therefore, we investigatedif OsNPR1 modulates the production of TrypPIs andHIPVs. Constitutive TrypPI levels in as-npr1 lines didnot differ from those in WT plants (Fig. 3B, C). However,when infested by SSB or LF, the levels of TrypPI activityin as-npr1 lines N6 and N7, measured 3 days afterthe start of herbivore infestation, were significantlyincreased compared to those in WT plants (Fig. 3B, C).

Volatiles emitted from WT and as-npr1 plants thatwere infested by SSB for 24 h were collected and ana-lyzed. The results show that SSB infestation significantlyenhanced the release of volatiles in both WT and as-npr1plants (Fig. 4, Table 1). While there was no obvious differ-ence in constitutive volatile-release between WT plantsand as-npr1 plants except for sesquithujene that washigher in as-npr1 lines than in WT plants, SSB-infestedplants of as-npr1 lines N6 or N7 emitted significantlyhigher amounts of volatiles than did identically treatedWT plants. The emissions of 14 compounds, including12 terpenoids, (E)-linalool oxide, linalool, α-copaene,sesquithujene, (E)-β-Caryophyllene, (E)-α-bergamotene,sesquisabinene A, α-curcumene, zingiberene, β-bisabolene, β-sesquiphellandrene and (E)-γ -bisabolene,and two unknown chemicals were significantlyincreased in as-npr1 lines (Fig. 4, Table 1).


A B

C D

E F

Fig. 2. Expression of OsNPR1, OsHI-LOX and OsACS2, and phytohormone levels in rice stems of as-npr1 lines and WT plants after they were infestedwith the rice striped stem borer (SSB). (A) Mean expression levels (+SE, n = 5) of OsNPR1 in stems of as-npr1 lines and WT plants at 8 h after SSBinfestation. (B) Mean SA levels (+SE, n = 5) in stems of as-npr1 lines and WT plants at 0 and 3 h after SSB infestation. (C) Mean JA levels (+SE, n = 5)in stems of as-npr1 lines and WT plants at 0 and 3 h after SSB infestation. (D) Mean expression levels (+SE, n = 5) of OsHI-LOX in stems of as-npr1lines and WT plants at 0, 0.5 and 1 h after SSB infestation. (E) Mean ET levels (+SE, n = 5) emitted from as-npr1 lines and WT plants at 0, 12 and24 h after SSB infestation. (F) Mean expression levels (+SE, n = 5) of OsACS2 in stems of as-npr1 lines and WT plants at 0, 0.5 and 1 h after SSBinfestation. Asterisks indicate significant differences between as-npr1 lines compared to WT plants at the same time point (*P < 0.05; **P < 0.01;Student’s t-test).

Antisense expression of OsNPR1 increases riceresistance to SSB

TrypPIs in rice have been reported to influence thelarval performance of SSB (Zhou et al. 2009). We thusasked whether silencing npr1 influences the growth ofSSB caterpillars. As expected, compared with larvaefeeding on WT plants, SSB caterpillars on as-npr1 linesgrew significantly less (Fig. 3A). The growth rates ofSSB larvae on N6 and N7 were only 47.5 and 68.3%,compared to WT controls.

Discussion

Here, we present evidence for the involvement ofOsNPR1 in the herbivore-mediated induction of JA andET pathways, and in the direct and indirect defenses of

rice. Several lines of evidence support this statement.First, although transcripts of OsNPR1 increase afterwounding, infestation by SSB and LF caterpillars,and treatment with JA and SA, wounding elicits theexpression of OsNPR1 quickly, whereas herbivoreinfestation elicits it more slowly (Fig. 1). Second, theantisense expression of OsNPR1 increases elicited levelsof JA and ET but not of SA (Fig. 2), which subsequentlyresults in increases in TrypPI activity (Fig. 3) andHIPVs (Fig. 4). These data suggest that OsNPR1 has animportant function in the herbivore-induced defenseresponses. Further studies will be required to elucidatehow OsNPR1 regulates the biosynthesis of JA and ET.

The basic function of NPR1 is twofold: The activationof SA-mediated SAR in the nucleus (Kinkema et al. 2000)and the regulation of the SA-mediated suppression of


A

B

C

Fig. 3. Resistance of as-npr1 lines and WT plants to the rice stripedstem borer (SSB). (A) Mean growth rates (+SE, n = 30) of individualsecond-instar SSB larvae 7 days after they fed on either WT plants oras-npr1 lines. (B) Mean levels (+SE, n = 5) of TrypPIs in stems of as-npr1lines and WT plants 3 days after plants were infested by SSB or keptnon-manipulated (Con). (C) Mean levels (+SE, n = 5) of TrypPIs in leavesof as-npr1 lines and WT plants 3 days after plants were infested bythe rice leaf folder (LF) or kept non-manipulated (Con). Letters indicatesignificant differences among different treatments (P < 0.05, Duncan’smultiple-range test); asterisks indicate significant differences betweenas-npr1 lines compared to WT plants (*P < 0.05; **P < 0.01, Student’st-test).

JA-dependent defenses in the cytosol (Stotz et al. 2002,Rayapuram and Baldwin 2007). The second aspectseems to differ between plant species. In Arabidopsis,NPR1 mediates the SA suppression of JA biosynthesis,and thus silencing NPR1 increases JA levels andJA-dependent defense when plants are infested bypathogens (Spoel et al. 2003). In N. attenuata, however,NPR1 inhibits the biosynthesis of SA that suppresses JAproduction, therefore leading to higher SA and lower JAlevels when plants are silenced with NPR1 (Rayapuramand Baldwin 2007). We found that antisense expressionof OsNPR1 enhances the levels of herbivore-induced

JA and JA-dependent defenses (Fig. 2). Therefore, therole of OsNPR1 in plant defense response is similar tothe role of AtNPR1 but not the role of NaNPR1. Thisfinding is consistent with the result that AtNPR1 is morehomologous to OsNPR1 than is NaNPR1 in evolutionand biochemistry (Rayapuram and Baldwin 2007, Maieret al. 2011), and with the data reported by Chern et al.(2001), who showed that rice had a NPR1-mediateddisease-resistance pathway similar to Arabidopsis.

In accordance with the previous results reportedby Yuan et al. (2007), transcript levels of OsNPR1are upregulated when plants are treated by SA or JA(Fig. 1). Interestingly, mechanical wounding enhancesthe mRNA levels of OsNPR1 quickly, whereas herbivoreinfestation elicits them more slowly (Fig. 1 B–D), muchlater than the time when the JA level in SSB-infestedplants reaches its maximum (3 h after SSB infestation)(Zhou et al. 2011). It has been well documented thatherbivore infestation elicits higher JA levels than doesmechanical wounding in many plant species, includingrice (Wu and Baldwin 2009, Kanno et al. 2011). Givenan obvious inhibitory role of OsNPR1 on JA and ETbiosynthesis (Fig. 2), it may be that rice plants perceivethe herbivore infestation and then inhibit the increasein mRNA levels of OsNPR1 at the early stage; thus,plants may prioritize JA- and ET-dependent defenseresponses against herbivores. The increase in expressionlevels of OsNPR1 at the later time points, such as 8h after herbivore infestation (Fig. 1D), which decreasesthe JA and ET levels, might be a regulation strategy ofrice plants, resulting in appropriate defense responsesaccording to the damage by the invader. Our previousstudies found that SSB infestation elicits a JA burst thatreaches a maximum at 3 h after infestation, subsidingto the control level at 8 h (Zhou et al. 2011), aphenomenon that corresponds to the expression ofOsNPR1, which reaches a maximum after 8 h ofinfestation (Fig. 1D). Therefore, rice plants may haveevolved to modulate the JA and ET burst by regulatingthe timing of OsNPR1 expression, which may result in aspecific and appropriate defense response.

Interestingly, we found that there was no differencein constitutive levels of JA and ET between WT plantsand as-npr1 lines, although the basal transcript levelsof OsNPR1 in as-npr1 lines were significantly lowerthan those in WT plants (Fig. 2). Moreover, although theexpression levels of OsNPR1 were altered at 8 h afterSSB infestation, the antisense expression of OsNPR1increased JA levels after 3 h of SSB infestation (Fig. 2).It has been reported that increases in transcript levelsof JA biosynthesis-related genes do not contribute tothe initial increase in endogenous JA in plants infestedby herbivores, but contribute to sustaining the JA burst


Fig. 4. Typical chromatograms obtained by headspace collections from non-manipulated plant (Con) and SSB-infested plants (for 24 h) (SSB) of WTplants and as-npr1 lines N6 and N7. Numbers represent chemicals that are the same as in Table 1.

(Ziegler et al. 2001, Halitschke and Baldwin 2005). Forthe ET production, there may be a similar mechanismat work: It has been reported in A. thaliana thatphosphorylation of AtACS2 and AtACS6 by AtMPK6will enhance the production of ET (Liu and Zhang2004). This may explain why lower constitutive levelsof OsNPR1 transcripts in as-npr1 lines did not influencethe basal levels of JA and ET, but they did increase theJA and ET levels when as-npr1 lines were infested byherbivores (Fig. 2). Consistent with the results found inthe Arabidopsis mutant npr1 (Spoel et al. 2003), wefound that antisense inhibition of OsNPR1 also resultedin increases in mRNA levels of OsHI-LOX, a 13-LOXgene that is involved in the production of herbivore-induced JA in rice (Zhou et al. 2009). This suggeststhat, like AtNPR1 (Spoel et al. 2003), OsNPR1 regulatesJA biosynthesis by mediating the transcript levels of JAbiosynthesis-related genes such as OsHI-LOX. Furtherstudies should be conducted to elucidate how OsNPR1modulates the expression levels of these genes.

In addition to enhancing JA biosynthesis, theantisense expression of OsNPR1 also increases theproduction of the herbivore-induced ET (Fig. 2E) andthe transcript levels of OsACS2, a gene encoding a1-aminocyclopropane-1-carboxylate (ACC) synthase(ACS) that is involved in ET biosynthesis (Lu et al.

2011). A similar result was reported by Pieterse et al.(1998), who found that the Arabidopsis mutant npr1releases higher ET levels than do WT plants wheninfected by pathogens; moreover, when plants weretreated with ACC, the levels of ET emitted from thenpr1 mutant were twofold higher than those fromWT plants, suggesting higher ACC oxidase activity inthe npr1 mutant than in WT plants. This shows thatET metabolism is also mediated by OsNPR1 via theregulation of ET biosynthesis-related genes.

Both JA and ET pathways have been reported to playan important role in mediating herbivore-induced ricedefense responses, including a positive regulation for theproduction of TrypPIs and HIPVs (Zhou et al. 2009, Luet al. 2011, Lu et al. 2011, Qi et al. 2011, Wang et al.2011). Furthermore, in rice, TrypPIs have been reportedto influence the performance of SSB (Zhou et al. 2009),and HIPVs protect plants from attack by herbivores byattracting the natural enemies of the herbivores (Lou etal., 2005a, 2005b, Qi et al. 2011). Thus, it is likelythat OsNPR1 negatively regulates levels of TrypPIs andHIPVs as well as rice resistance to the chewing herbivoreSSB (Figs 3 and 4) via its influence on the biosynthesisof JA and ET (Fig. 2).

In summary, we propose that OsNPR1 is anearly responding gene in herbivore-induced defense


Table 1. Volatile compounds emitted by WT plants and as-npr1 lines N6 and N7 by health plants (Con) and plants infested by striped stem borer(SSB) for 24 h. Data represent the mean amount (% of IS peak area) of five replications. Letters in a same row indicate significant differences amongtreatments (P < 0.05, Duncan’s multiple range test).

No Chemical WT Con N6 Con N7 Con WT SSB N6 SSB N7 SSB

1 2-Heptanone 0.76 ± 0.10 b 4.64 ± 0.69 c 2.01 ± 0.51 b 51.55 ± 9.10 a 70.82 ± 21.21 a 58.62 ± 6.31 a2 2-Heptanol 1.23 ± 0.20 b 2.25 ± 0.29 b 1.82 ± 0.20 b 29.83 ± 4.11 a 41.94 ± 10.82 a 40.80 ± 4.69 a3 α-Thujene 0.47 ± 0.11 b 0.54 ± 0.11 b 0.81 ± 0.15 b 3.09 ± 0.49 ab 4.21 ± 0.49 a 3.93 ± 0.92 a4 α-Pinene 2.10 ± 0.80 b 0.87 ± 0.12 b 1.47 ± 0.07 b 4.52 ± 0.72 a 5.21 ± 0.76 a 5.70 ± 1.17 a5 (+)-Limonene 5.13 ± 1.10 d 9.51 ± 1.54 cd 14.00 ± 1.29 bcd 60.80 ± 8.72 abc 75.28 ± 9.46 a 59.33 ± 13.14 ab6 (E)-Linalool oxide 0.26 ± 0.07 e 0.73 ± 0.19 d 0.59 ± 0.11 de 2.97 ± 0.36 c 21.80 ± 1.14 a 9.44 ± 0.57 b7 Linalool 1.10 ± 0.39 c 1.06 ± 0.48 c 1.24 ± 0.53 c 19.33 ± 2.10 b 141.96 ± 10.36 a 101.74 ± 12.79 a8 Unknown 2.98 ± 0.48 d 3.92 ± 0.52 cd 5.93 ± 0.69 abc 4.75 ± 0.45 bcd 6.97 ± 0.43 ab 9.51 ± 2.53 a9 Methyl salicylate 3.11 ± 0.29 b 4.17 ± 0.73 b 6.18 ± 0.69 ab 5.03 ± 0.49 ab 8.56 ± 0.79 a 6.79 ± 2.64 ab

10 α-Copaene nd nd nd 5.27 ± 1.53 b 23.34 ± 4.24 a 19.17 ± 4.40 a11 Sesquithujene 0.51 ± 0.09 d 1.72 ± 0.25 c 2.41 ± 0.21 bc 3.43 ± 0.53 b 11.66 ± 1.86 a 9.03 ± 1.80 a12 (E)-β-Caryophyllene 0.88 ± 0.10 d 1.84 ± 0.28 c 1.39 ± 0.33 cd 3.71 ± 0.43 b 10.74 ± 1.24 a 10.91 ± 1.78 a13 (E)-α-Bergamotene 0.52 ± 0.24 c 0.34 ± 0.12 c 0.44 ± 0.07 c 6.43 ± 1.75 b 37.18 ± 7.84 a 25.86 ± 4.75 a14 Sesquisabinene A 0.44 ± 0.02 d 1.42 ± 0.29 cd 1.12 ± 0.11 c 7.16 ± 1.74 b 38.73 ± 8.14 a 26.33 ± 4.68 a15 Unknown 0.37 ± 0.06 c 0.60 ± 0.09 c 2.24 ± 0.30 b 2.49 ± 0.68 b 13.15 ± 2.76 a 9.81 ± 2.03 a16 α-Curcumene nd nd nd 2.14 ± 0.55 b 14.66 ± 3.17 a 9.79 ± 1.90 a17 Zingiberene 12.31 ± 1.35 c 15.03 ± 1.88 bc 11.31 ± 1.07 c 21.51 ± 3.18 b 73.89 ± 12.99 a 51.71 ± 8.90 a18 β-Bisabolene 0.47 ± 0.08 c 0.53 ± 0.14 c 0.57 ± 0.07 c 9.56 ± 0.47 b 39.98 ± 8.04 a 27.64 ± 4.58 a19 β-Sesquiphellandrene 0.41 ± 0.05 c 1.32 ± 0.38 c 0.64 ± 0.18 c 10.61 ± 2.79 b 58.15 ± 15.07 a 46.25 ± 7.37 a20 (E)-γ -Bisabolene 0.23 ± 0.03 c 0.60 ± 0.08 c 1.40 ± 0.19 c 3.96 ± 0.98 b 22.96 ± 7.77 a 17.49 ± 2.31 a

responses in rice (Fig. 5). When attacked by herbivores,plants perceive the damage and inhibit increasesin the activity of OsNPR1 at the early stage, whichsubsequently increases the production of JA and ET byenhancing the activity of JA and ET biosynthesis-relatedenzymes, such as LOX and ACS. Together, the activatedJA and ET signaling pathways result in an effective andspecific herbivore-induced direct and indirect defenseresponse. At the late stage of herbivore infestation,increased OsNPR1 activity impairs the biosynthesis ofJA and ET, which buffers the response and keeps thedefensive investment at an appropriate level.

Acknowledgements – We thank Jiancai Li, Tongfang Zhangand Xiaopeng Wang for their invaluable assistance withthe experiments, and thank Emily Wheeler for editorialassistance. The study was jointly sponsored by the NationalBasic Research Program of China (2010CB126200), theInnovation Research Team Program of the National NaturalScience Foundation of China (31021003), the NationalNatural Science Foundation of China (31071695), and theNational Program of Transgenic Variety Development ofChina (2011ZX08009-003-001).

References

Cao H, Bowling SA, Gordon AS, Dong XN (1994)Characterization of an Arabidopsis mutant that isnonresponsive to inducers of systemicacquired-resistance. Plant Cell 6: 1583–1592

Fig. 5. Preliminary model summarizing how OsNPR1 may regulateherbivore-induced signaling pathways and defenses in rice. Whenattacked by herbivores, plants perceive the damage and inhibit in theactivity of OsNPR1 at the early stage (ES), which subsequently increasesthe production of JA and ET by enhancing the activity of JA and ETbiosynthesis-related enzymes, such as LOX and ACS. The activatedJA and ET signaling pathways then produce an effective and specificdirect and indirect defense response. At the late stage (LS) of herbivoreinfestation, increased OsNPR1 activity impairs the biosynthesis of JA andET, which buffers the response and keeps the defensive investment atan appropriate level.

Cao H, Glazebrook J, Clarke JD, Volko S, Dong XN (1997)The Arabidopsis NPR1 gene that controls systemicacquired resistance encodes a novel protein containingankyrin repeats. Cell 88: 57–63

Cao H, Li X, Dong XN (1998) Generation ofbroad-spectrum disease resistance by overexpression ofan essential regulatory gene in systemic acquiredresistance. Proc Natl Acad Sci USA 95: 6531–6536


Cheng JA, He JH (1996) Rice Insect Pests. ChinaAgriculture Press, Beijing

Chern MS, Fitzgerald HA, Yadav RC, Canlas PE, Dong XN,Ronald PC (2001) Evidence for a disease-resistancepathway in rice similar to the NPR1-mediated signalingpathway in Arabidopsis. Plant J 27: 101–113

Chern M, Fitzgerald HA, Canlas PE, Navarre DA, RonaldPC (2005) Overexpression of a rice NPR1 homologleads to constitutive activation of defense response andhypersensitivity to light. Mol Plant Microbe Interact 18:511–520

Cui J, Jander G, Racki LR, Kim PD, Pierce NE, Ausubel FM(2002) Signals involved in Arabidopsis resistance totrichoplusia ni caterpillars induced by virulent andavirulent strains of the phytopathogen Pseudomonassyringae. Plant Physiol 129: 551–564

Despres C, DeLong C, Glaze S, Liu E, Fobert PR (2000)The Arabidopsis NPR1/NIM1 protein enhances the DNAbinding activity of a subgroup of the TGA family of bZIPtranscription factors. Plant Cell 12: 279–290

Fan WH, Dong XN (2002) In vivo interaction betweennpr1 and transcription factor TGA2 leads to salicylicacid-mediated gene activation in Arabidopsis. Plant Cell14: 1377–1389

Feng JX, Cao L, Li J, Duan CJ, Luo XM, Le N, Wei HH,Liang SJ, Chu CC, Pan QH, Tang JL (2011) Involvementof OsNPR1/NH1 in rice basal resistance to blast fungusMagnaporthe oryzae. Eur J Plant Pathol 131: 221–235

Fitzgerald HA, Chern MS, Navarre R, Ronald PC (2004)Overexpression of (At)NPR1 in rice leads to a BTH- andenvironment-induced lesion-mimic/cell deathphenotype. Mol Plant Microbe Interact 17: 140–151

Girling RD, Madison R, Hassall M, Poppy GM, Turner JG(2008) Investigations into plant biochemicalwound-response pathways involved in the production ofaphid-induced plant volatiles. J Exp Bot 59: 3077–3085

Halitschke R, Baldwin IT (2005) Jasmonates and relatedcompounds in plant-insect interactions. J Plant GrowthRegul 23: 238–245

Isikawa K, Yoshimura K, Harada K, Fukusaki E, Ogawa T,Tamoi M, Shigeoka S (2010) AtNUDX6, anADP-Ribose/NADH pyrophosphohydrolase inArabidopsis, positively regulates NPR1-dependentsalicylic acid signaling. Plant Physiol 152: 2000–2012

Kanno H, Hasegawa M, Kodama O (2011) Accumulationof salicylic acid, jasmonic acid and phytoalexins in rice,Oryza sativa, infested by the white-backed planthopper,Sogatella furcifera (Hemiptera: Delphacidae). ApplEntomol Zool 47: 27–34

Kinkema M, Fan WH, Dong XN (2000) Nuclearlocalization of NPR1 is required for activation of PRgene expression. Plant Cell 12: 2339–2350

Koornneef A, Pieterse CMJ (2008) Cross talk in defensesignaling. Plant Physiol 146: 839–844

Leon-Reyes A, Spoel SH, De Lange ES, Abe H, KobayashiM, Tsuda S, Millenaar FF, Welschen RA, Ritsema T,Pieterse CM (2009) Ethylene modulates the role ofNONEXPRESSOR OF PATHOGENESIS-RELATEDGENES1 in cross talk between salicylate and jasmonatesignaling. Plant Physiol 149: 1797–1809

Li J, Brader G, Palva ET (2004) The WRKY70 transcriptionfactor: a node of convergence for jasmonate-mediatedand salicylate-mediated signals in plant defense. PlantCell 16: 319–331

Lindermayr C, Sell S, Muller B, Leister D, Durner J (2010)Redox regulation of NPR1-TGA1 system of Arabidopsisthaliana by nitric oxide. Plant Cell 22: 2894–2907

Liu YD, Zhang SQ (2004) Phosphorylation of1-aminocyclopropane-1-carboxylic acid synthase byMPK6, a stress-responsive mitogen-activated proteinkinase, induces ethylene biosynthesis in Arabidopsis.Plant Cell 16: 3386–3399

Lou YG, Baldwin IT (2003) Manduca sexta recognition andresistance among allopolyploid Nicotiana host plants.Proc Natl Acad Sci USA 100: 14581–14586

Lou YG, Du MH, Turlings TCJ, Cheng JA, Shan WF(2005a) Exogenous application of jasmonic acid inducesvolatile emissions in rice and enhances parasitism ofNilaparvata lugens eggs by the parasitoid Anagrusnilaparvatae. J Chem Ecol 31: 1985–2002

Lou YG, Ma B, Cheng JA (2005b) Attraction of theparasitoid Anagrus nilaparvatae to rice volatiles inducedby the rice brown planthopper Nilaparvata lugens. JChem Ecol 31: 2357–2372

Lou YG, Hua XY, Turlings TCJ, Cheng JA, Chen XX, Ye GY(2006) Differences in induced volatile emissions amongrice varieties result in differential attraction andparasitism of Nilaparvata lugens eggs by the parasitoidAnagrus nilaparvatae in the field. J Chem Ecol 32:2375–2387

Lu YJ, Wang X, Lou YG, Cheng JA (2006) Role of ethylenesignaling in the production of rice volatiles induced bythe rice brown planthopper Nilaparvata lugens. Chin SciBull 51: 2457–2465

Lu J, Ju HP, Zhou GX, Zhu CS, Erb M, Wang XP, Wang P,Lou YG (2011) An EAR-motif-containing ERFtranscription factor affects herbivore-induced signaling,defense and resistance in rice. Plant J 68: 583–596

Maier F, Zwicker S, Huckelhoven A, Meißner M, Funk J,Fitzner AJP, Pfitzner UM (2011) NONEXPRESSOR OFPATHOGENESIS-RELATED PROTEINS1 (NPR1) andsome NPR1-related proteins are sensitive to salicylicacid. Mol Plant Pathol 12: 73–91

Meur G, Budatha M, Srinivasan T, Rajesh Kumar KR, DuttaGupta A, Kirti PB (2008) Constitutive expression ofArabidopsis NPR1 confers enhanced resistance to theearly instars of Spodoptera litura in transgenic tobacco. JPlant Physiol 133: 765–775


Mewis I, Tokuhisa JG, Schultz JC, Appel HM, Ulrichs C,Gershenzon J (2006) Gene expression and glucosinolateaccumulation in Arabidopsis thaliana in response togeneralist and specialist herbivores of different feedingguilds and the role of defense signaling pathways.Phytochemistry 67: 2450–2462

Mou Z, Fan W, Dong X (2003) Inducers of plant systemicacquired resistance regulate NPR1 function throughredox changes. Cell 113: 935–944

Parkhi V, Kumar V, Campbell LAM, Bell AA, Shah J,Rathore KS (2010) Resistance against various fungalpathogens and reniform nematode in transgenic cottonplants expressing Arabidopsis NPR1. Transgenic Res 19:959–975

Petersen M, Brodersen P, Naested H, Andreasson E,Lindhart U, Johansen B, Nielsen HB, Lacy M, Austin MJ,Parker JE, Sharma SB, Klessig DF, Martienssen R,Mattsson O, Jensen AB, Mundy J (2000) ArabidopsisMAP kinase 4 negatively regulates systemic acquiredresistance. Cell 103: 1111–1120

Pieterse CMJ, van Loon LC (2004) NPR1: the spider in theweb of induced resistance signaling pathways. CurrOpin Plant Biol 7: 456–464

Pieterse CMJ, van Wees SCM, van Pelt JA, Knoester M,Laan R, Gerrits H, Weisbeek PJ, van Loon LC (1998) Anovel signaling pathway control induced systemicresistance in Arabidopsis. Plant Cell 10: 1571–1580

Qi JF, Zhou GX, Yang LJ, Erb M, Lu YH, Sun XL, Cheng JA,Lou YG (2011) The chloroplast-localizedphospholipases D α4 and α5 regulateherbivore-induced direct and indirect defenses in rice.Plant Physiol 157: 1987–1999

Rayapuram C, Baldwin IT (2007) Increased SA inNPR1-silenced plants antagonizes JA and JA-dependentdirect and indirect defenses in herbivore-attackedNicotiana attenuata in nature. Plant J 52: 700–715

Shah J (2003) The salicylic acid loop in plant defense. CurrOpin Plant Biol 6: 365–371

Shimono M, Sugano S, Nakayama A, Jiang CJ, Ono K, TokiS, Takatsuji H (2007) Rice WRKY45 plays a crucial rolein benzothiadiazole-inducible blast resistance. PlantCell 19: 2064–2076

Spoel SH, Koornneef A, Claessens SM, Korzelius JP, vanPelt JA, Mueller MJ, Buchala AJ, Metraux JP, Brown R,Kazan K, van Loon LC, Dong XN, Pieterse CM (2003)NPR1 modulates cross-talk between salicylate- andjasmonate-dependent defense pathways through a novelfunction in the cytosol. Plant Cell 15: 760–770

Spoel SH, Mou ZL, Tada Y, Spivey NW, Genschik P, DongXN (2009) Proteasome-mediated turnover of thetranscription coactivator NPR1 plays dual roles inregulating plant immunity. Cell 137: 860–872

Stotz HU, Koch T, Biedermann A, Weniger K, Boland W,Mitchell-Olds T (2002) Evidence for regulation ofresistance in Arabidopsis to Egyptian cotton worm by

salicylic and jasmonic acid signaling pathways. Planta214: 648–652

Sugano S, Jiang CJ, Miyazawa SI, Masumoto C, Yazawa K,Hayashi N, Shimono M, Nakayama A, Miyao M,Takatsuji H (2010) Role of OsNPR1 in rice defenseprogram as revealed by genomewide expressionanalysis. Plant Mol Biol 74: 549–562

van Dam NM, Horn M, Mares M, Baldwin IT (2001)Ontogeny constrains systemic protease inhibitorresponse in Nicotiana attenuata. J Chem Ecol 27:547–568

Wally O, Jayaraj J, Punja ZK (2009) Broad-spectrumdisease resistance to necrotrophic and biotrophicpathogens in transgenic carrots (Daucus carota L.)expressing an Arabidopsis NPR1 gene. Planta 231:131–141

Wang X, Hu LC, Zhou GX, Cheng JA, Lou YG (2011)Salicylic acid and ethylene signaling pathways areinvolved in production of rice trypsin proteinaseinhibitors induced by the leaf folder Cnaphalocrocismedinalis (Guenee). Chin Sci Bull 56: 2351–2358

Wu JQ, Baldwin IT (2009) Herbivory-induced signalling inplants: perception and action. Plant Cell Environ 32:1161–1174

Yoshida S, Forno DA, Cock JH, Gomez KA (1976)Laboratory Manual for Physiological Studies of Rice, 3rdEdn. The International Rice Research Institute, Manila

Yuan YX, Zhong SH, Li Q, Zhu ZR, Lou YG, Wang LY,Wang JJ, Wang MY, Li QL, Yang DL, He ZH (2007)Functional analysis of rice NPR1-like genes reveals thatOsNPR1/NH1 is the rice orthologue conferring diseaseresistance with enhanced herbivore susceptibility. PlantBiotechnol J 5: 313–324

Zhou GX, Qi JF, Ren N, Cheng JA, Erb M, Mao BZ, Lou YG(2009) Silencing OsHI-LOX makes rice more susceptibleto chewing herbivores, but enhances resistance to aphloem feeder. Plant J 60: 638–648

Zhou GX, Wang X, Yan F, Wang X, Li R, Cheng JA, LouYG (2011) Genome-wide transcriptional changes anddefense-related chemical profiling of rice in response toinfestation by the rice striped stem borer Chilosuppressalis. Physiol Plant 143: 21–40

Ziegler J, Keinanen M, Baldwin IT (2001)Herbivore-induced allene oxide synthase transcripts andjasmonic acid in Nicotiana attenuate. Phytochemistry58: 729–738

Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Table S1. Primers and probes used for QRT-PCR of targetgenes.


Fig. S1. cDNA sequence alignment of OsNPR1, OsNPR2and OsNPR3. Sequence used for OsNPR1 antisensetransformation is underlined. The blue shading indicatesthe bases that are identical in the three genes.

Fig. S2. Rice transformation vector pCAMBIA-NPR1(13.3 kb) with Hyg and GUS as plant selectable markergenes.

Fig. S3. DNA gel-blot analysis and growth phenotypes ofas-npr1 lines and WT plants. (A) DNA gel-blot analysis ofWT line and four as-npr1 T2 lines (N6, N7, N9 and N11).Genomic DNA was digested with EcoRI or XbaI. The blotwas hybridized with a probe (about 0.7 kb) specific for

reporter gene GUS. Hybridization was created using theDIG High Prime DNA Labeling and Detection Starter KitII (Roche). All four as-npr1 lines have a single insertionof the transgene. (B–D) Growth phenotypes of as-npr1lines and WT plants at 10-days old (B), tillering stage (C)and heading stage (D).

Please note: Wiley-Blackwell are not responsible forthe content or functionality of any supporting materialssupplied by the authors. Any queries (other than missingmaterial) should be directed to the corresponding authorfor the article.

Edited by R. Terauchi


Documents

OsNPR1 negatively regulates herbivore-induced JA and ethylene signaling and plant resistance to a chewing herbivore in rice