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Keywords:

  • rice;
  • ethylene responsive factor;
  • herbivore resistance;
  • defense-related signaling;
  • mitogen-activated protein kinase;
  • WRKY

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Ethylene responsive factors (ERFs) are a large family of plant-specific transcription factors that are involved in the regulation of plant development and stress responses. However, little to nothing is known about their role in herbivore-induced defense. We discovered a nucleus-localized ERF gene in rice (Oryza sativa), OsERF3, that was rapidly up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis. Antisense and over-expression of OsERF3 revealed that it positively affects transcript levels of two mitogen-activated protein kinases (MAPKs) and two WRKY genes as well as concentrations of jasmonate (JA), salicylate (SA) and the activity of trypsin protease inhibitors (TrypPIs). OsERF3 was also found to mediate the resistance of rice to SSB. On the other hand, OsERF3 was slightly suppressed by the rice brown planthopper (BPH) Nilaparvata lugens (Stål) and increased susceptibility to this piercing sucking insect, possibly by suppressing H2O2 biosynthesis. We propose that OsERF3 affects early components of herbivore-induced defense responses by suppressing MAPK repressors and modulating JA, SA, ethylene and H2O2 pathways as well as plant resistance. Our results also illustrate that OsERF3 acts as a central switch that gears the plant’s metabolism towards an appropriate response to chewing or piercing/sucking insects.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In nature, plants are constantly threatened by herbivorous insects. This has led to the evolution of plant defenses, many of which are induced specifically after herbivore attack (Heil and Baldwin, 2002; Bruce and Pickett, 2007; Kessler and Halitschke, 2007; Maffei et al., 2007; Howe and Jander, 2008). The induction of defense starts with the recognition of specific molecular patterns from herbivores (Turlings et al., 2000; Schmelz et al., 2009; Wu and Baldwin, 2010), followed by the activation of a complex regulatory network involving mitogen-activated protein kinase (MAPK) cascades, jasmonate (JA), salicylate (SA) and ethylene signaling, the expression of defense-related genes and the production of defensive chemicals (Bostock, 2005; Browse and Howe, 2008; Howe and Jander, 2008; Wu and Baldwin, 2010). Although transcription factors (TFs) have long been assumed to play an important role in regulating herbivore-induced plant defense responses (Schittko et al., 2001; Halitschke et al., 2003), only a few TFs have been characterized in that respect (Dombrecht et al., 2007; Wang et al., 2007; Skibbe et al., 2008). The best-studied TFs involved in plant–insect interactions are MYC2 and the WRKYs (Dombrecht et al., 2007; Skibbe et al., 2008). AtMYC2 in Arabidopsis thaliana was reported to locate downstream of JA and to regulate JA-mediated plant herbivore resistance (Dombrecht et al., 2007). In Nicotiana attenuata, two WRKY genes, NaWRKY3 and NaWRKY6, were found to modulate elicited JA and JA–Ile/–Leu biosynthesis, and, subsequently, herbivore-induced defense responses (Skibbe et al., 2008). It still remains unclear whether other TFs are important for insect resistance and to what extent TFs regulate resistance in monocotyledonous plants.

Ethylene responsive factors (ERFs) are a large family of plant-specific stress-responsive TFs that have not been investigated as possible players in plant–insect interactions. In 1995, Ohme-Takagi and Shinshi first reported that four ethylene responsive DNA-binding proteins from Nicotiana tabacum had a conserved domain. These proteins were then named ethylene responsive element-binding proteins (EREBPs) or ERFs, although they were not always responsive to ethylene (Oñate-Sánchez and Singh, 2002; Tournier et al., 2003). The ERFs include a conserved 58–59 amino acid DNA-binding domain (designated as the AP2/ERF domain) and specifically bind to GCC cis-elements in the promoter of their target genes (Ohmetakagi and Shinshi, 1995; Fujimoto et al., 2000; Nakano et al., 2006). In A. thaliana and rice (Oryza sativa), there are 122 and 139 ERF genes belonging to 12 and 15 groups (Nakano et al., 2006). The ERFs have been reported to play important roles in manipulating biological processes related to plant growth and development as well as plant responses to various biotic and abiotic stresses, including pathogen infection (Gutterson and Reuber, 2004; Nakano et al., 2006; Xu et al., 2008). Most ERFs act as activators that positively regulate the transcript levels of their target genes, whereas a few of them, such as some members in group VIII, act as repressors (Orozco-Cárdenas et al., 2001; Kazan, 2006; Nakano et al., 2006). In contrast to the ERF activators, the ERF repressors contain a conserved (L/F)DLN(L/F)xP sequence, also called the ERF-associated amphiphilic repression (EAR) motif, in their C-terminal regions (Ohta et al., 2001). Previous studies revealed that ERF repressors are activated by pathogen infection, mechanical wounding and treatment with different signals, such as JA, ethylene and abscisic acid (ABA), and generally act as negative regulators of plant responses to stresses (Fujimoto et al., 2000; McGrath et al., 2005; Song et al., 2005; Yang et al., 2005). For instance, McGrath et al. (2005) found that AtERF4 is a negative regulator of JA-responsive defense gene expression, and over-expression of AtERF4 caused the plants to become more susceptible to the necrotrophic fungal pathogen Fusarium oxysporum. In addition, over-expressing AtERF7 in A. thaliana reduced the sensitivity of guard cells to ABA and increased transpirational water loss, whereas silencing AtERF7 increased sensitivity to ABA (Song et al., 2005). In summary, while ERFs, including repressor-type ERFs, seem to be central to many plant regulatory processes, including plant disease resistance, their role in herbivore-induced defense responses remains almost entirely unclear.

In this study we isolated a rice repressor type ERF gene, OsERF3 (Ohta et al., 2001), which was induced by herbivore infestation, and elucidated its role in herbivore-induced plant defense responses. Transcriptional analysis revealed that OsERF3 is rapidly induced by caterpillar feeding and mechanical wounding, but only slowly elicited by treatment with JA or SA. OsERF3 positively regulates expression levels of two MAPK genes and two WRKY genes, JA, SA and ethylene biosynthesis, and trypsin protease inhibitor (TrypPI) activity, but negatively modulates H2O2 biosynthesis. Over-expression or silencing of OsERF3 demonstrates that the gene is a positive regulator of plant resistance to a chewing herbivore, the rice striped stem borer (SSB) Chilo suppressalis, and a susceptibility factor against a phloem-feeder, the rice brown planthopper (BPH) Nilaparvata lugens (Stål), both of which are important insect pests in rice. In summary, we propose that OsERF3 is a central early herbivore-responsive gene that influences a number of defense-related signaling pathways, including MAPK cascades as well as JA, SA and ethylene signaling. Also, OsERF3 seems to be an important switch that regulates defense responses of rice against chewing and piercing/sucking insects.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Isolation and characterization of OsERF3

Using suppressive subtractive hybridization (SSH), we screened rice plants for herbivore-induced transcripts. Using this technique, we identified a clone that showed similarity to an ERF transcription factor. The full-length cDNA of the cloned SSB-induced OsERF, including an open reading frame (ORF) of 708 bp, was then obtained by RT-PCR (Figure S1 in Supporting Information). Blast analysis showed that the sequence was highly similar (99.8%) to that of the previously identified OsERF3 (TIGR ID Os01g58420), suggesting that the isolated OsERF is OsERF3.

To clarify the subcellular localization of OsERF3, we constructed an OsERF3:EGFP fusion gene, driven by a CaMV 35S promoter (Figure S2a), and transiently expressed the construct in N. tabacum leaves. Fluorescence analysis indicated that OsERF3 exclusively localizes in the nucleus (Figure 1).

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Figure 1.  Localization of OsERF3 in Nicotiana tabacum cells. Nicotiana tabacum cells were transformed with OsERF3:EGFP and EGFP. After incubation for 36 h, the transformed cells were observed under a confocal microscope. The photographs were taken in UV light, visible light and in combination (overlay), respectively.

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Quantitative real-time polymerase chain reaction (QRT-PCR) analysis revealed low constitutive expression of OsERF3, while mechanical wounding, SSB and rice leaf folder (LF) Cnaphalocrocis medinalis caterpillar infestation resulted in a rapid increase (<1 h) in transcript levels (Figure 2a–c). A BPH infestation did not induce OsERF3, but slightly repressed OsERF3 1 h after attack (Figure 2d). Treatment with JA and SA also increased the expression of OsERF3, but the elicitation was slower than after wounding, SSB or LF attack (Figure 2e, f). These results led to the hypothesis that OsERF3 might be an early responsive gene in plant–herbivore interactions and may regulate herbivore-induced defense responses in rice.

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Figure 2.  Expression of OsERF3 in rice stems or leaves after different treatments. Mean transcript levels (+SE, = 5) of OsERF3 in rice stems that were mechanically wounded (a), infested by rice stripped stem borer (SSB) (b), rice brown planthopper (BPH) (d), or treated with jasmonic acid (JA) (e) or salicylic acid (SA) (f). Mean transcript levels (+SE, = 5) of OsERF3 in rice leaves that were infested by rice leaf folder (LF) (c). BUF, buffer; C, non-manipulation; non-infested, plant stems were covered with an empty glass cage. Transcript levels were analyzed by quantitative RT-PCR. Asterisks indicate significant differences in transcript levels between treatments and controls (*< 0.05; **P < 0.01, Student’s t-tests).

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Over-expressing and silencing of OsERF3

To determine the role of OsERF3 in herbivore-induced defense responses, we constructed pCAMBIA-1301 transformation vectors carrying reverse fragments (∼ 0.6-kb coding regions) or a forward full-length ORF of OsERF3 (Figure S2b, c), and generated transgenic rice plants using Agrobacterium tumefaciens-mediated transformation. By GUS staining and hygromycin resistance selection, we obtained four T2 homozygous lines, including two OsERF3 silenced lines (as-erf lines: as-1 and as-2) and two OsERF3 over-expression lines (oe-ERF lines: oe-1 and oe-2), all of which contain a single T-DNA insertion (Figure S3b, d). Transcriptional analysis showed that SSB-induced transcript levels of OsERF3 in as-1 and as-2 lines were about 42.50 and 53.16% of those in wild-type (WT) plants at 1 h after SSB infestation. In contrast, the transcript levels were significantly increased in oe-1 (5.15-fold) and oe-2 lines (8.91-fold), compared with WT plants at 1 h after SSB infestation (Figure S3a, c).

Silencing OsERF3 did not influence the phenotype of rice plants, while over-expression of OsERF3 during the seedling stage led to a slight inhibition of root elongation – 10 days after sowing, the root length in the oe-1 and oe-2 was decreased by about 7.48 and 12.02% compared with WT plants (Figure S4a, c). This influence disappeared at later developmental stages (Figure S4b, d).

OsERF3 regulates transcript levels of two MAPKs and two WRKYs

Mitogen-activated protein kinase cascades play an essential role in mediating the responses of plants to various stresses (Romeis, 2001; Zhang and Klessig, 2001; Rodriguez et al., 2010). Wu et al. (2007) demonstrated that two N. attenuata MAPK genes, NaWIPK and NaSIPK, are upstream signaling components regulating herbivore-induced JA, SA and ethylene biosynthesis. Here, we investigated if expression of OsMPK3 and OsMPK6, the homologs of NaWIPK and NaSIPK, and OsMEK3, the homolog of AtMEK3 that can phosphorylate AtMPK6 (Takahashi et al., 2007), in transgenic plants differed from those in WT plants. As shown in Figure 3b, transcript levels of OsMPK3 in WT plants increased rapidly (in less than 15 min) when infested by SSB and peaked at 90 min after herbivore infestation. Compared with WT plants, over-expressing OsERF3 significantly enhanced constitutive and herbivore-induced expression levels of OsMPK3, and silencing OsERF3 resulted in lower transcript levels of OsMPK3. Unlike OsMPK3, OsMPK6 was not elicited by SSB infestation, and its transcript levels were similar in WT plants and plants with altered OsERF3 expression (Figure S5). Although SSB attack did not enhance levels of OsMEK3 mRNA in WT plants, the over-expression of OsERF3 led to higher transcript levels of OsMEK3 compared with WT plants, and silencing OsERF3 resulted in lower levels (Figure 3a). These results suggest that OsERF3 positively regulates the transcript levels of OsMPK3 and OsMEK3, but not OsMPK6.

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Figure 3.  OsERF3 regulates transcript levels of mitogen-activated protein kinases (MAPKs) and WRKYs. Mean transcript levels (+SE, = 5) of OsMEK3 (a), OsMPK3 (b), OsWRKY53 (c) and OsWRKY70 (d) in rice stems of as-erf and oe-ERF plants, and wild-type (WT) plants that were individually infested by a third-instar rice stripped stem borer (SSB) larva. Transcript levels were analyzed by quantitative RT-PCR. Asterisks indicate significant differences in as-erf and oe-ERF lines compared with WT plants (*< 0.05, Student’s t-tests).

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In plants, some WRKYs locate downstream of MAPK cascades and participate in plant immune responses (Asai et al., 2002; Qiu et al., 2008). Skibbe et al. (2008) demonstrated that NaWRKY3 and NaWRKY6 mediate herbivore-induced specific defenses in N. attenuata via activation of JA signaling. We therefore investigated the transcriptional levels of two WRKY genes, OsWRKY 53, the homolog of NaWRKY6, and OsWRKY 70, the homolog of AtWRKY33, that regulate plant resistance to necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola (Zheng et al., 2006), in WT plants, and as-erf and oe-ERF lines. While both genes were rapidly up-regulated in SSB-infested WT plants, their transcript levels were reduced in as-erf lines and further increased in oe-ERF lines (Figure 3c, d), indicating that OsERF3 regulates the expression of OsWRKY53 and OsWRKY70.

OsERF3 mediates herbivore-induced JA, SA, ethylene and H2O2 biosynthesis

The JA signaling pathway has been reported to play a central role in plant defense response in many plant species, including rice (Chung et al., 2008; Howe and Jander, 2008; Stork et al., 2009; Zhou et al., 2009). Therefore, we determined whether OsERF3 influences JA biosynthesis. The results show that basal JA levels were similar between as-erf lines and WT plants, whereas JA levels in as-erf lines were significantly decreased compared with WT plants upon SSB attack; the levels of JA in the as-erf lines, as-1 and as-2, were only 59.20 and 67.48, and 72.36 and 66.83% of those in WT plants 1.5 h and 3 h after SSB infestation, respectively (Figure 4a). Consistent with this finding, over-expression of OsERF3 significantly increased constitutive and SSB-induced JA levels; the levels of JA in the oe-ERF lines, oe-1 and oe-2, were 1.16 and 1.33, and 1.14 and 1.25-fold higher than those in WT plants 1.5 h and 3 h after SSB infestation, respectively (Figure 4a).

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Figure 4.  OsERF3 positively mediates jasmonate (JA) biosynthesis. (a) Mean levels (+SE, = 5) of JA in as-erf and oe-ERF lines, and wild-type (WT) plants that were individually infested by a third-instar rice stripped stem borer (SSB) larva. (b)–(d) Mean transcript levels (+SE, = 5) of JA biosynthesis-related genes OsHI-LOX (b), OsAOS1 (c) and OsOPR1 (d) in as-erf and oe-ERF lines, and WT plants that were individually infested by a third-instar SSB larva. Asterisks indicate significant differences in as-erf and oe-ERF lines compared with WT plants (*< 0.05, Student’s t-tests).

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Zhou et al. (2009) demonstrated that OsHI-LOX, a homolog of AtLOX2 encoding a 13-lipoxygenase, is involved in herbivore-induced JA biosynthesis in rice. Thus, we examined the changes in transcript levels of OsHI-LOX and two other JA biosynthesis-related genes, OsAOS1, a putative allene oxide synthase (AOS) gene and OsOPR1, a homolog of AtOPR1 encoding another enzyme for JA biosynthesis (Yara et al., 2008), in the transgenic lines and WT plants after infestation by SSB. The results show that SSB infestation in WT plants resulted in a rapid accumulation of mRNA for the three genes, with a peak at 90 min after infestation (Figure 4b-d). Compared with WT plants, silencing OsERF3 significantly decreased transcript levels of OsHI-LOX and OsOPR1 while over-expression of OsERF3 increased mRNA accumulation of the two genes (Figure 4b, d), suggesting a positive regulation of OsERF3 for OsHI-LOX and OsOPR1. Unlike OsHI-LOX and OsOPR1, the expression of OsAOS1 in transgenic plants was similar to those in WT plants (Figure 4c). Significant differences were only found at 0 min between oe-ERF lines and WT plants and at 90 min between as-erf lines and WT plants (Figure 4c). The data suggest that OsERF3 regulates mRNA levels of OsHI-LOX and OsOPR1, but not OsAOS1.

Salicylate and ethylene signaling are also important for manipulating herbivore-induced plant defense responses (Arimura et al., 2005; Kessler and Halitschke, 2007; Howe and Jander, 2008). In rice, previous studies have demonstrated that herbivore infestation induces the production of SA and ethylene (Wang, 2006; Zhou et al., 2009). To determine whether OsERF3 is involved in SA and ethylene biosynthesis, we analyzed SA and ethylene levels in the different lines. The WT plants and oe-ERF lines had similar constitutive and induced SA levels, whereas basal and induced levels of SA in as-erf lines were only about half of those in WT plants (Figure 5a). As shown in Figure 5b, the accumulation of ethylene was similar in as-erf lines and WT plants, but the levels were significantly elevated in oe-ERF lines.

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Figure 5.  OsERF3 mediates rice stripped stem borer (SSB)-induced salicylic acid (SA), ethylene and H2O2 accumulation. (a), (b) Mean levels (+SE, = 5) of SA (a) and ethylene (b) in as-erf and oe-ERF lines, and wild-type (WT) plants that were individually infested by a third-instar SSB larva. (c) Mean concentrations (+SE, = 5) of H2O2 in as-erf and oe-ERF lines, and WT plants that were individually infested by 12 rice brown planthopper (BPH) female adults. (d) Mean transcript levels (+SE, = 5) of OsACS2 in as-erf and oe-ERF lines, and WT plants that were individually infested by a third-instar SSB larva. Asterisks indicate significant differences in as-erf and oe-ERF lines compared with WT plants (*< 0.05, Student’s t-tests).

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1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), which converts S-adenosyl-l-methionine (S-AdoMet) to ACC, is a key enzyme for ethylene biosynthesis (Lin et al., 2009). As the ACS gene responsible for ethylene production has not been identified in rice, we screened ACS homologs and found that one of them, OsACS2, mediates herbivore-induced ethylene production (Figures S6 and S7). We therefore measured the expression pattern of OsACS2 in the different OsERF3 genotypes. In accordance with the measured ethylene production, the basal and induced transcript levels of OsACS2 in oe-ERF lines were increased, whereas the levels of OsACS2 mRNA in as-erf lines were similar to those in WT plants, except for the 90-min harvest, when mRNA levels of OsACS2 in the as-1 line were lower than in WT plants (Figure 5d).

In addition to JA, SA and ethylene, H2O2 is another important signaling molecule that plays a vital role in induced plant defense (Orozco-Cárdenas et al., 2001; Lou and Baldwin, 2006). Our previous results show that H2O2 signaling is likely to be involved in the resistance of rice to BPH (Zhou et al., 2009). Therefore, we measured H2O2 concentrations in transgenic lines and WT plants when they were infested by BPH. The results revealed that BPH-elicited H2O2 accumulation was significantly increased in as-erf lines compared with WT plants, but obviously decreased in oe-ERF lines (Figure 5c). Together, our data demonstrate that OsERF3 positively modulates JA, SA and ethylene biosynthesis, but reduces H2O2.

Mutants with impaired JA or ethylene biosynthesis do not influence the levels of OsERF3 transcripts

Our results show that JA and SA slowly elicited the expression of OsERF3 (Figure 2). Moreover, methyl jasmonate (MeJA), did not induce the accumulation of OsERF3 up to 4 h after treatment (Figure S8). To further explore the notion that OsERF3 may be an upstream component that regulates the biosynthesis of JA, SA and ethylene, we investigated the expression of OsERF3 in transgenic plants with impaired JA and ethylene signaling. We used an antisense OsHI-LOX line (as-lox), which produces only 50% of the JA levels compared with WT plants when infested by SSB (Zhou et al., 2009), and an antisense-ACS2 line (as-acs), which produces significantly less SSB-elicited ethylene than WT plants (Figure S7). The experiments revealed that the levels of constitutive and induced OsERF3 transcripts in as-lox and as-acs plants were the same as those in WT plants over the first 60 min (Figure S9). Taken together with the results above, this indicates that OsERF3 is induced upstream of JA and ethylene signaling in rice.

OsERF3 positively regulates TrypPI production and resistance to SSB

In rice, JA, SA and ethylene signaling have an important role in plant defenses against herbivores (Lu et al., 2006; Zhou et al., 2009). Thus, we assessed the performance of SSB on transgenic lines and WT plants to determine whether OsERF3 mediates resistance to rice herbivores. The SSB caterpillars gained more mass on as-erf lines as-1 and as-2 than on WT plants (Figure 6a). By day 12, the masses of caterpillars that fed on as-1 and as-2 were 1.35- and 1.35-fold higher, respectively, than those that fed on WT plants. In contrast, the larval weight of SSB caterpillars that fed on oe-ERF lines oe-1 and oe-2 were decreased by 15.85% and 19.61%, respectively, compared with those fed on WT plants (Figure 6a). Moreover, as-erf lines were more severely damaged by SSB than WT plants, whereas oe-ERF lines were less damaged (Figures 6c and S10). Trypsin protease inhibitors are important direct defense proteins against SSB in rice (Zhou et al., 2009). In accordance with the SSB bioassays, TrypPI activity was suppressed in as-erf lines but enhanced in oe-ERF lines compared with those in WT plants (Figure 6b). These results show that OsERF3 positively regulates TrypPI activity and the resistance of rice to SSB.

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Figure 6.  OsERF3 positively regulates rice resistance to rice stripped stem borer (SSB). (a) Mean larval mass (+SE, n = 60) of SSB that fed on as-erf lines, oe-ERF lines or wild-type (WT) plants for 12 days. (b) Mean trypsin protease inhibitor (TrypPI) activities (+SE, = 5) in as-erf and oe-ERF lines, and WT plants that were individually infested by a third-instar SSB larva for 3 days. (c) Damaged phenotypes of as-erf lines, oe-ERF lines and WT plants that were individually infested by a third-instar SSB larva for 8 or 12 days (= 10). Asterisks indicate significant differences in as-erf and oe-ERF lines compared with WT plants (*< 0.05; **< 0.01, Student’s t-tests).

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OsERF3 negatively regulates resistance to BPH

When as-erf lines and WT plants were exposed to a BPH colony, BPH adult females preferred to feed on WT plants rather than as-erf lines (Figure 7a, b). Similarly, BPH adult females laid more eggs on WT plants than either as-erf line – the numbers of eggs on the as-erf lines as-1 and as-2 were only 30.58 and 15.58% of the number laid on WT plants, respectively (Figure 7a, b, insets). In line with these findings, BPH adult females were found more often on oe-ERF lines than on WT plants when both of the rice genotypes were offered to BPH simultaneously, and laid more eggs on the former than on the latter (Figure 7c, d). Moreover, BPH nymphs fed on as-erf lines had lower survival rates than those fed on WT plants, especially nymphs who fed on as-erf line as-1 (Figure 7e); by contrast, BPH nymphs on oe-ERF lines had higher survival rates (Figure 7f). These experiments suggest that OsERF3 negatively regulates the resistance of rice to BPH.

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Figure 7.  OsERF3 negatively regulates rice resistance to rice brown planthopper (BPH). (a)–(d) Mean number of adult female BPH per plant (+SE, = 6) on pairs of plants (wild type (WT) versus as-1, as-2, oe-1 and oe-2, respectively), 1–48 h after pairs were exposed. Inserts: mean percentage (+SE, n = 6) of BPH eggs per plant on pairs of plants as stated above, 48 h after the release of BPH. (e), (f) Mean survival rate (+SE, = 6) of BPH nymphs that fed on as-erf lines, oe-ERF lines or WT plants 1–12 days after the start of feeding. Asterisks indicate significant differences in as-erf and oe-ERF compared with WT plants (*< 0.05; **< 0.01, Student’s t-tests).

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Exogenous application of MeJA restores induced TrypPI levels and SSB resistance in as-erf lines

The as-erf lines had lower induced JA and SA levels and higher H2O2 levels than WT plants, whereas in the oe-ERF lines, JA and ethylene levels were enhanced and H2O2 levels were suppressed (Figures 4 and 5). To determine whether the differences in JA production were sufficient to explain the changes in TrypPI production and the resistance of rice to SSB, we compared the differences in induced TrypPI levels and larval performance of SSB between as-erf lines and WT plants after MeJA treatment. Treatment with MeJA and SSB attenuated the difference in TrypPI levels between WT plants and as-erf lines (Figure 8a). Moreover, SSB larvae that fed on MeJA-treated as-erf lines gained a similar amount of weight as those fed on equally treated WT plants (Figure 8b). The fact that the reduction in plant resistance to SSB and elicited accumulation of TrypPI in as-erf lines can be completely restored by exogenous application of MeJA further indicates that jasmonate signaling is an important component of the OsERF3-mediated changes in plant defense.

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Figure 8.  Exogenous application of methyl jasmonate (MeJA) restores trypsin protease inhibitor (TrypPI) levels and rice resistance to rice stripped stem borer (SSB) in as-erf lines. (a) Mean activities (+SE, = 5) of TrypPIs in as-erf and oe-ERF lines, and wild-type (WT) plants that were individually treated with 100 μg of MeJA in 20 μl of lanolin paste (MeJA) or with 20 μl of pure lanolin (insert) for 24 h, followed by SSB feeding for 3 days. (b) Mean larval mass (+SE, = 60) of SSB 12 days after they fed on as-erf and oe-ERF lines, and WT plants that were individually treated with 100 μg of MeJA in 20 μl of lanolin paste (MeJA) or with 20 μl of pure lanolin (insert) for 24 h. Asterisks indicate significant differences in as-erf and oe-ERF lines compared with WT plants (*< 0.05; **< 0.01, Student’s t-tests).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

ERF transcription factors as central components of plant defense responses

Our results show that ERF transcription factors can play an important role in shaping herbivore-induced defense responses of plants. Specifically, OsERF3 appears to be a central early switch in induced defense in rice. Several lines of evidence are presented here that support this claim. First, OsERF3 is rapidly induced by infestation with SSB and LF, whereas JA, MeJA or SA treatment elicits its expression much more slowly (Figures 2 and S8). Second, over-expressing or silencing OsERF3 modulates transcript levels of defense-related genes, including MAPKs and WRKYs, defense-related signal molecules (JA, SA, ethylene and H2O2) and defense compounds, such as TrypPIs (Figures 3–6). The fact that not all investigated transcripts and signals in as-erf or oe-ERF lines, such as OsMEK3, OsWRKY53, OsWRKY70, SA and ethylene, were modulated (Figures 3–6) suggests an involvement of other factors in their regulation. Third, compared with WT plants, mutations in JA or ethylene biosynthesis did not influence levels of OsERF3 transcripts in plants (up to 1 h after infestation, Figure S9). Finally, OsERF3 transcripts were significantly accumulated by 5 min after mechanical wounding, which preceded expression of MAPKs, WRKYs and signal biosynthesis-related genes such as OsMPK3, OsWRKY53, OsWRKY70, OsHI-LOX, OsAOS1, OsOPR1 and OsACS2 (Figure S11). These data suggest that OsERF3 is an early wound- and herbivore-responsive gene that is probably located upstream of defense-related signaling pathways. Alternatively, OsERF3 may regulate early components of defense signaling via positive and/or negative feedback loops. Future research involving genotypes that are depleted in JA or impaired in MAPK signaling will be helpful to assess this possibility.

Possible mechanisms of OsERF3 as a positive regulator of gene expression

While many repressor-type ERFs, such as AtERF4 and AtERF7, are known to negatively regulate transcript levels of defense-related genes and subsequent plant stress tolerance (Fujimoto et al., 2000; McGrath et al., 2005; Song et al., 2005; Yang et al., 2005), we found that all of the tested defense-related genes, as well as the signaling molecules JA, SA and ethylene, were positively regulated by OsERF3 in rice (Figures 3-7). This observation was found to be in agreement with studies of ERF repressors in tomato (SlERF3) and sugarcane (SodERF3), although whether they positively modulate defense genes remains unclear (Trujillo et al., 2008; Chen et al., 2009). The positive effect of OsERF3 and other repressor-type ERFs on defense gene expression suggests that OsERF3 might function as an inhibitor that suppresses one or more repressors of defense responses. In this case, when plants are infested by herbivores enhanced OsERF3 activity would rapidly suppress the repressors and remove their inhibition, thereby resulting in the activation of defense responses (Figure 9). A similar regulatory model has been put forward in another example of an EAR-motif-containing transcriptional regulator, C2H2-(Cys2/His2) type zinc-finger proteins (Ciftci-Yilmaz et al., 2007). OsERF3 activity may also be enhanced via phosphorylation by a protein kinase. This mechanism has been observed in Arabidopsis, where a protein kinase, PKS3, phosphorylates AtERF7, a member of group VIII, thereby enhancing the DNA-binding and/or repression activity of AtERF7 (Song et al., 2005). Nevertheless, the identification of proteins interacting with OsERF3 will be required to test these hypotheses.

image

Figure 9.  Preliminary model summarizing how OsERF3 may regulate herbivore-induced signaling pathways and defenses. Plants recognize signals from wounding and herbivore infestation and quickly activate OsERF3. OsERF3 rapidly suppresses the expression of one or more putative repressors (R) that inhibit mitogen-activated protein kinase (MAPK) cascades and/or WRKYs, and thereby activates MAPKs, such as OsMPK3 and OsMEK3, and/or WRKYs. Activating MAPKs and WRKYs enhances transcript levels of other transcription factors, including other WRKYs, and defense signal biosynthesis-related genes, such as OsHI-LOX, OsAOS1, OsOPR1 and OsACS2, which, taken together, increase the concentrations of jasmonic acid (JA), salicylic acid (SA) and ethylene (ET). Meanwhile, activating MAPKs keeps H2O2 at a certain level by inhibiting its production. The resulting signaling profile will shape the defense response of rice. Here, JA, possibly together with ethylene, induces the production of trypsin protease inhibitors and other defense chemicals, which directly defend against rice striped stem borer (SSB); H2O2 signaling may be mainly related to the resistance of rice to rice brown planthopper (BPH).

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OsERF3 and its influence on MAPKs, WRKYs and phytohormones

As an early responsive element, OsERF3 influences many aspects of a plant’s defense response. Our findings suggest that the different elements act in a cascade starting from OsERF3-induced MAPK signaling, followed by WRKY regulation and phytohormone production. In plants, MAPK cascades are pivotal signaling pathways that modulate stress responses. Their activation occurs mainly via post-translational and transcriptional regulation (Kim and Zhang, 2004; Seo et al., 2007; Wu et al., 2007). Here we show that OsERF3 positively regulates transcriptional expression of at least two MAPKs (Figure 3). Similar results have been reported in A. thaliana, where plants over-expressing AtERF104 displayed higher basal levels of AtMKK4, a component located upstream of AtMPK6, compared with Col-0 (WT) plants (Bethke et al., 2009). Mitogen-activated protein kinases are also known to be upstream regulators for some WRKY transcripts (Pandey and Somssich, 2009), which we found to be differentially regulated by OsERF3 as well (Figure 3). In N. attenuata, the transcript accumulation of NaWRKY6, the homolog of OsWRKY53, was found to be modulated by two MAPKs, NaWIPK and NaSIPK (Wu et al., 2007). In A. thaliana, Pseudomonas syringae infection activates MPK4 of the MPK4/MKS1/WRKY33 complex, which subsequently phosphorylates MKS1 and then results in the release of WRKY33 (Qiu et al., 2008). We propose here that OsERF3 regulates transcript levels of the two WRKY genes via modulation of MAPKs, such as OsMPK3. Whether OsERF3 can also directly influence the accumulation of the two WRKY transcripts, by suppressing repressors of WRKYs, for example, needs to be addressed in further studies.

Mitogen-activated protein kinases and WRKYs have been shown mediate the biosynthesis of JA, SA, ethylene and H2O2. In N. attenuata for example, NaSIPK and NaWIPK positively regulate JA, SA and JA–Ile biosynthesis, and NaSIPK also positively regulates ethylene production (Wu et al., 2007). Skibbe et al. (2008) demonstrated that silencing NaWRKY6 (the homolog of OsWRKY53) and NaWRKY3 reduced Manduca sexta-elicited JA levels. In A. thaliana, AtMPK6 phosphorylates AtACS2 and AtACS6, which subsequently elevates ACS activity and the production of ethylene (Liu and Zhang, 2004). Recently, a tomato ERF activator, SlERF2, has been reported to control ethylene production by regulating transcriptional expression of SlACO3 (Zhang et al., 2009). Increasing evidence also indicates that MAPKs control the production of reactive oxygen species (ROS) (Ichimura et al., 2006; Zong et al., 2009; Ning et al., 2010). For example, in A. thaliana, the MEKK1–MPK4 cascade was reported to negatively regulate H2O2 production (Nakagami et al., 2006), suggesting that this cascade may include a feedback loop for H2O2 production and signaling. Given the strong effect of OsERF3 on MAPK cascades and WRKYs found here, OsERF3 regulation of JA, SA, ethylene and H2O2 levels may be achieved mainly through MAPK cascades and WRKYs. Whether OsERF3 can also regulate phytohormone production directly remains to be elucidated.

OsERF3 as a central switch for induced defenses against herbivores with different feeding styles

Our experiments demonstrate that OsERF3 positively regulates TrypPI activity and the resistance of rice to the chewing herbivore SSB (Figure 6), possibly via JA signaling (Figure 8). On the other hand, this transcription factor appears to be a negative regulator of resistance against BPH, a homopteran phloem feeder of rice (Figure 7). This finding is consistent with our previous results showing that as-lox plants, which had lower elicited JA levels, were susceptible to SSB but more resistant to BPH (Zhou et al., 2009). The increased resistance to BPH in as-lox plants was proposed to be related to its higher BPH-induced H2O2 levels, which together with higher elicited SA, induce a hypersensitive response (HR)-like cell death that inhibits BPH feeding (Zhou et al., 2009). On as-erf plants, we did not observe a HR-like cell death phenomenon when they were infested by BPH (data not shown), which is probably related to the lower induced SA levels of these plants. The observation that both as-lox and as-erf plants were more resistant to BPH, and the finding that as-erf lines had lower induced SA and higher H2O2 levels (Figure 5a, c) while as-lox plants have both higher SA and H2O2 levels (Zhou et al., 2009), indicates that the H2O2 signaling pathway might play an important role in the resistance of rice to BPH. It is noteworthy in this context that BPH does not only perform better on ERF3-expressing plants, but that it also prefers to settle on them, an effect which may stem from a reduction of deterrents or an induction of attractive or arrestant compounds.

Our results demonstrate that OsERF3 may act as a switch that gears the plant’s response in the right direction depending on the type of attacker. We found that OsERF3 is activated upon attack by chewing herbivores (Figure 2), which improves the plant’s resistance against the attacker (Figure 6), but is slightly suppressed upon attack by a piercing sucking insect (Figure 7). Thus, this transcription factor appears to contribute to the plant’s capacity to respond specifically to the type of herbivore it encounters. It is also interesting to speculate about whether OsERF3 regulation can lead to negative interaction between defenses against chewing and piercing sucking insects beyond the often evoked JA/SA antagonism (Spoel et al., 2003). In this context, our findings may contribute to elucidate the mechanisms behind ecologically important, plant-mediated interactions between herbivores (Poelman et al., 2009).

In summary, we demonstrate that OsERF3 is an early responsive gene in herbivore-induced defense responses in rice. It regulates JA, SA, ethylene and H2O2 signaling pathways by via MAPK cascades and WRKYs, which taken together results in an effective and specific herbivore-induced defense response (Figure 9). Our study provides a compelling example of how a single gene can act as a switch for the activation of multiple herbivore-induced signaling pathways and herbivore resistance.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant growth

The rice genotypes used in this study were Xiushui 110 WT and transgenic lines of as-erf, oe-ERF, as-acs (see below) and as-lox (Zhou et al., 2009). Pre-germinated seeds of the different lines were cultured in plastic bottles (diameter 8 cm, height 10 cm) in a greenhouse (28 ± 2°C, 14 h light, 10 h dark). Ten-day-old seedlings were transferred to 20-L hydroponic boxes with a rice nutrient solution (Yoshida et al., 1976). After 40 days, seedlings were transferred to individual 500-ml hydroponic plastic pots. Plants were used for experiments 4–5 days after transplanting.

Insects

Colonies of SSB, LF and BPH were originally obtained from rice fields in Hangzhou, China, and maintained on Xianyou 63 (a rice variety that is susceptible to SSB and BPH) in a controlled climate chamber at 26 ± 2°C, with a 12-h light phase and 80% relative humidity.

Isolation and characterization of OsERF3 cDNA

The full-length cDNA of OsERF3 was obtained by RT-PCR from total RNA isolated from WT plants infested by a SSB larva for 24 h. The primers ERF-F1 (5′-CCCAAACCCAACCTCCCA-3′) and ERF-R1 (5′-CGTCAGCTAACCCATCCTG-3′) were designed based on the sequence of the rice OsERF3 (TIGR ID Os01g58420), which showed high homology with the partial sequence of the OsERF transcript that was cloned by SSH. The PCR-amplified fragments were cloned into the pMD19-T vector (TaKaRa, http://www.takara-bio.com/) and sequenced.

Generation and characterization of transgenic plants

The full-length cDNA sequence and a 572 bp fragment of OsERF3 were inserted into the pCAMBIA-1301 transformation vector, yielding an over expression and an antisense construct. Both vectors were inserted into the rice variety Xiushui 110 using A. tumefaciens-mediated transformation. Rice transformation, screening of the homozygous T2 plants and identification of the number of insertions followed the same method as described in Zhou et al. (2009). Two T2 homozygous lines (as-1 and as-2) of as-erf and two lines (oe-1 and oe-2) of oe-ERF, each harboring a single insertion (Figure S3), were used in subsequent experiments.

Similarly, we cloned a 421-bp fragment of OsACS2 (TIGR ID Os04g48850; Figure S6) and inserted it into the pCAMBIA-1301 transformation vector in an antisense orientation (Wang, 2009). Using the same procedure as stated above, we obtained two T2 homozygous lines with antisense inhibition of OsACS (as-acs line 1 and line 30), each with a single insertion (Figure S7).

Plant treatments

For mechanical wounding, plant stems (lower part, about 2 cm long) were individually pierced 200 times using a needle (W). Non-manipulated plants were used as controls (C). For SSB treatment, plants were infested using a third-instar larva of SSB that had been starved for 2 h. Controls (C) were not manipulated. For LF treatment, plants were individually infested using a third-instar larva of LF that had been starved for 2 h and then placed on leaves at node 4 (the youngest fully expanded leaf was defined as leaf node 1). Non-manipulated plants were used as controls (C). For BPH treatment, plants were individually infested with 12 female adults of BPH that were confined in a glass cage (diameter 4 cm, height 8 cm, with 48 small holes, diameter 0.8 mm). Plants with an empty cage were used as controls (non-infested). For SA or JA treatment, plants were individually sprayed with 2 ml of SA (70 μg ml−1) or JA solution (100 μg ml−1) in 50 mm sodium phosphate buffer. Controls (BUF) were sprayed with 2 ml of the buffer. For MeJA treatment, plant stems were individually treated with 100 μg of MeJA in 20 μl of lanolin paste. Controls (Lanolin) were similarly treated with 20 μl of pure lanolin.

Subcellular localization

The full-length ORF without a stop codon of OsERF3 cDNA was cloned into the pEGFP vector (BD Biosciences, http://www.bdbiosciences.com/) to fuse it with EGFP. The fusion gene, OsERF3:EGFP, was inserted into pCAMBIA1301, yielding a transformation vector (Figure S2a). This vector was used for transient transformation of N. tabacum leaves as described by Wroblewski et al. (2005). Fluorescence analysis was performed as described by Zhou et al. (2009).

Quantitative real-time PCR

For QRT-PCR analysis, five independent biological samples were used. Total RNA was isolated using the SV Total RNA Isolation System (Promega, http://www.promega.com/), following the manufacturer’s instructions. One microgram of each total RNA sample was reverse transcribed using the PrimeScript® RT-PCR Kit (TaKaRa). The QRT-PCR assay was performed on an ABI PRISM 7500 sequence detection system (Applied Biosystems, http://www.appliedbiosystems.com/) using a Premix Ex Taq® Kit (TaKaRa). A rice actin gene OsACT (TIGR ID Os03g50885) was used as an internal standard to normalize cDNA concentrations. The primers and probes used for QRT-PCR for all tested genes are provided in Table S1.

Jasmonate and SA analysis

Plants of the different genotypes were randomly assigned to SSB and control treatments. Stems were harvested at 0, 1.5 and 3 h after SSB treatment, and JA and SA levels were analyzed by gas chromatography (GC)-MS using labeled internal standards as described previously (Lou and Baldwin, 2003). Each treatment at each time interval was replicated five times.

Ethylene analysis

Plants of the different genotypes were randomly assigned to SSB and control treatments, and covered with sealed glass cylinders (diameter 4 cm, height 50 cm). Ethylene production was determined using a method described by Lu et al. (2006). Each treatment at each time interval was replicated five times.

Quantification of hydrogen peroxide

Wild-type plants and plants of as-erf and oe-ERF lines were randomly assigned to BPH and control treatment. Leaf sheaths were harvested at 0, 8 and 24 h after treatment. Each treatment at each time interval was replicated five times. The H2O2 concentrations were determined using Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen, http://www.invitrogen.com/) as described previously by Lou and Baldwin (2006).

Analysis of TrypPI activity

Plant stems (0.12–0.15 g per sample) were harvested 3 days after the start of treatment. 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.

Herbivore resistance experiments

Three freshly hatched SSB larvae were allowed to feed on transgenic (as-1, as-2, oe-1 and oe-2) and WT plants. Twenty plants were used for each line. Larval mass (to an accuracy of 0.1 mg) was measured 12 days after the start of the experiment.

To investigate the colonization and oviposition behavior of BPH, pots with two plants (a transgenic plant versus a WT plant) were confined with glass cylinders (diameter 4 cm, height 8 cm, with 48 small holes, diameter 0.8 mm). Each cylinder received 12 gravid BPH females. The number of BPH on each plant was counted at 1, 2, 4, 8, 12, 24, 36 and 48 h after the release of BPH, and then BPH were removed and the eggs on each plant were counted under a microscope. The experiments were replicated six times. The survival rates of BPH nymphs on WT and transgenic plants were also determined. For this, plant stems were individually covered with a glass cylinders, into which 10 BPH neonates were released. Every day, the numbers of surviving BPH on each plant was recorded until 12 days after the release of the herbivores. The experiment was replicated six times.

To determine the differences in the tolerance of plants to herbivore attack, the different genotypes were individually infested with individual SSB third-instar larva. Every day, the damage levels of plants were checked and photographs were taken.

Data analysis

Differences in herbivore performance, expression levels of genes and herbivore-induced JA, SA, ethylene and H2O2 levels on different treatments, lines or treatment times were determined by analysis of variance (Student’s t-tests for comparing two treatments). All tests were carried out with Statistica (Statistica, SAS Institute Inc., http://www.sas.com/).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Guilan Dong for invaluable assistance with the experiments. Abbie Ferrieri provided helpful comments on an earlier version of the manuscript. The study was jointly sponsored by the National Basic Research Program of China (2010CB126200), the Innovation Research Team Program of the National Natural Science Foundation of China (31021003), the National Natural Science Foundation of China (31071695; 30871644), the Natural Science Foundation of Zhejiang Province (D3080282) and the earmarked fund for Modern Agro-industry Technology Research System.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Nucleotide sequence and the deduced amino acid sequence of the cloned OsERF. The conserved ERF domain (Dark gray shading) and the EAR motif (underlined) were shown. Red letters indicate the nucleotides different from OsERF3.

Figure S2. Transformation vectors were used in this study.

Figure S3.OsERF3 expression levels and DNA gel-blot analysis of as-erf and oe-ERF lines and WT plants. (a, c) Mean transcript levels (+SE) of OsERF3 in as-erf and oe-ERF lines, and WT plants that were individually infested by a third-instar SSB larva. Transcript levels were analyzed by QRT-PCR. Asterisks indicate significant differences in as-erf and oe-ERF lines compared to WT plants (*, P  <  0.05; **, P  <  0.01, Student’s t-tests). (b, d) DNA Gel-Blot analysis of as-erf and oe-ERF lines, and WT plants. Genomic DNA was digested with EcoR I or Xba I. The blot was hybridized with a probe specific for reporter gene gus. All transgenic lines have a single insertion of the transgene.

Figure S4. Growth phenotypes of as-erf and oe-ERF lines and WT plants. (a) 10-days-old seedlings. (b) Plants at tillering stage. (c) Mean root length (+SE) of 10  days-old seedlings of WT plants and plants of as-erf and oe-ERF lines. Asterisks indicate significant differences in as-erf and oe-ERF lines compared to WT plants (*, P  <  0.05; **, P  <  0.01, Student’s t-tests). (d) Plants at heading stage.

Figure S5. Expression levels of OsMPK6 in as-erf and oe-ERF lines and WT plants. Mean transcript levels (+SE) of OsMEK6 transcript levels in as-erf and oe-ERF lines, and WT plants that were individually infested by a third-instar SSB larva. Transcript levels were analyzed by QRT-PCR. Asterisks indicate significant differences in as-erf and oe-ERF lines compared to WT plants (*, P  <  0.05, Student’s t-tests).

Figure S6. Nucleotide sequence and the deduced amino acid sequence of OsACS2.

Figure S7. OsACS2 mediates herbivore-induced ethylene production in rice.

(a) Mean transcript levels (+SE) of OsACS2 in two as-acs lines (line 1 and line 30) and WT Plants that were individually infested by a third-instar SSB larva for 3  h. Transcript levels were analyzed by QRT-PCR. Asterisks indicate significant differences in as-acs lines compared to WT plants (*, P  <  0.05; **, P  <  0.01, Student’s t-tests).

(b) DNA Gel-Blot analysis of as-acs lines and WT plants. Genomic DNA was digested with EcoR I (E) or Xba I (X). The blot was hybridized with a probe specific for reporter gene gus. All transgenic lines have a single insertion of the transgene.

(c) Mean levels (+SE) of ethylene in as-acs lines and WT plants that were individually infested by a third-instar SSB larva. Asterisks indicate significant differences in as-acs lines compared to WT plants (*, P  <  0.05; **, P  <  0.01, Student’s t-tests).

Figure S8. Mean transcript levels (+SE) of OsERF3 in rice stems of WT plants that were treated with 100  μg of MeJA in 20  μl of lanolin paste (MeJA) or with 20  μl of pure lanolin (C). Transcript levels were analyzed by QRT-PCR.

Figure S9. Levels of OsERF3 transcripts in WT plants and as-lox and as-acs lines. Plants were individually infested by a third-instar SSB larva.

Figure S10. Mean plant mass (+SE) of as-erf lines, oe-ERF lines and WT plants at 0, 8 or 12  days after they were individually infested by a third-instar SSB larva.

Figure S11. Expression levels of OsERF3 and other related genes in WT plants.

Mean transcript levels (+SE) of OsERF3 (a), OsMPK3 (b), OsWRKY53 (c), OsWRKY70 (d), OsHI-LOX (e), OsAOS1 (f), OsOPR1 (g) and OsACS2 (h) in WT plants that were mechanically wounded.

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

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