The jasmonic acid (JA) pathway plays a central role in plant defense responses against insects. Some phloem-feeding insects also induce the salicylic acid (SA) pathway, thereby suppressing the plant’s JA response. These phenomena have been well studied in dicotyledonous plants, but little is known about them in monocotyledons. We cloned a chloroplast-localized type 2 13-lipoxygenase gene of rice, OsHI-LOX, whose transcripts were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis and the rice brown planthopper (BPH) Niaparvata lugens, as well as by mechanical wounding and treatment with JA. Antisense expression of OsHI-LOX (as-lox) reduced SSB- or BPH-induced JA and trypsin protease inhibitor (TrypPI) levels, improved the larval performance of SBB as well as that of the rice leaf folder (LF) Cnaphalocrocis medinalis, and increased the damage caused by SSB and LF larvae. In contrast, BPH, a phloem-feeding herbivore, showed a preference for settling and ovipositing on WT plants, on which they consumed more and survived better than on as-lox plants. The enhanced resistance of as-lox plants to BPH infestation correlated with higher levels of BPH-induced H2O2 and SA, as well as with increased hypersensitive response-like cell death. These results imply that OsHI-LOX is involved in herbivore-induced JA biosynthesis, and plays contrasting roles in controlling rice resistance to chewing and phloem-feeding herbivores. The observation that suppression of JA activity results in increased resistance to an insect indicates that revision of the generalized plant defense models in monocotyledons is required, and may help develop novel strategies to protect rice against insect pests.
The biosynthesis of jasmonates starts with the release of linolenic acid from chloroplast membranes. Linolenic acid is then oxygenated via 13-lipoxygenase (13-LOX) into hydroperoxy polyunsaturated fatty acid, which is the common substrate for at least seven pathways (Feussner and Wasternack, 2002). Based on the positional specificity of the dioxygen insertion, plant LOXs are grouped into 9-LOXs and 13-LOXs, and, based on their sequence similarities, the latter can be further classified into two sub-families (types 1 and 2) (Feussner and Wasternack, 2002). Recent studies suggest that, in most cases, specific 13-LOX isoforms in the type 2 sub-family, which catalyze the dioxygenation of fatty acid at the C13 position and possess a chloroplast transit peptide, are involved in JA biosynthesis (see, for example, León et al., 2002). Several type 2 13-LOX genes have been identified in plants, including AtLOX2 in Arabidopsis thaliana (Bell et al., 1995), NaLOX3 in Nicotiana attenuata (Halitschke and Baldwin, 2003), TomloxD in Solanum lycopersicum (tomato) (Heitz et al., 1997) and LOX2:Hv:1 in Hordeum vulgare (barley) (Sharma et al., 2006). In rice, little is known about LOX isoforms belonging to this family (Peng et al., 1994; Schaffrath et al., 2000; Zabbai et al., 2004; Wang et al., 2008b). Furthermore, nothing is known about the relative contribution of JA-mediated defenses to resistance against phloem feeders and chewing herbivores in monocotyledonous plants, despite the fact that important rice pests include both feeding guilds (Cheng and He, 1996).
In the present study, we isolated an herbivore-induced rice type 2 13-LOX, designated OsHI-LOX, and analyzed its induced expression patterns and subcellular localization. To elucidate the role of OsHI-LOX in herbivore-induced defense responses, we silenced the activity of OsHI-LOX by antisense expression. The levels of JA, green leaf volatiles (GLVs) and trypsin protease inhibitors (TrypPIs) were measured in antisense OsHI-LOX (as-lox) lines and wild-type (WT) plants. To obtain insight into the ecological role of OsHI-LOX across feeding guilds, we examined the performance on these plants of two lepidopteran herbivores, the striped stem borer (SSB, Chilo suppressalis) and the rice leaf folder (LF, Cnaphalocrocis medinalis), and one phloem-feeding herbivore, rice brown planthopper (BPH, Niaparvata lugens), all of which are important insect pests in rice. Because BPH infestation induced a stronger hypersensitive response-like cell death in as-lox plants than in WT plants, we also determined the levels of SA and H2O2 in BPH-infested as-lox and WT plants.
Isolation, characterization and subcellular localization of rice OsHI-LOX
OsHI-LOX full-length cDNA, including an open reading frame of 2772 bp, a 5′ UTR of 14 bp and a 3′ UTR of 84 bp, was obtained by RT-PCR. Its deduced amino acid sequence revealed that OsHI-LOX encodes a protein of 924 amino acids, including a chloroplast transit peptide (Figure S1), with a calculated Mr of 102.82 kDa and a theoretical pI of 6.117. Sequence alignment revealed high similarity (98%) to a previously identified rice LOX gene OsRLL (Figure S2) (Peng et al., 1994). A homology search against the National Center for Biotechnology Information (NCBI) rice nucleotide database showed that both genes share high sequence identity along the same putative coding region on chromosome 8 (contig NC_008401, 25 205 188–25 246 731). This suggests that OsHI-LOX is allelic to OsRLL; their differences are attributed to their isolation from different rice varieties. In the phylogenetic tree of plant lipoxygenases constructed here (Figure S3), we classified the LOX proteins into two groups: group I enzymes carry a putative chloroplast transit peptide and all of them are 13-LOXs; group II enzymes have no transit peptide and have 9- or 13-LOX activity. OsHI-LOX belongs to group I (Figure S3), together with OsRCI-1, AtLOX2 and NaLOX3, whose sequence similarities to OsHI-LOX were 47.6, 52.2 and 45.5%, respectively (Figure S2). Thus, OsHI-LOX, a putative 13-LOX, may be localized to chloroplasts and be involved in herbivore-induced defense responses in rice.
A subcellular localization assay using a OsHI-LOX::GFP fusion gene, driven by the CaMV 35S promoter, showed that the autofluorescence emitted from chloroplasts (red, Figure 1a, panel I) completely overlapped (yellow, Figure 1a, panel III) with the fluorescence emitted from the fusion protein (green, Figure 1a, panel II), suggesting that OsHI-LOX localizes to chloroplasts.
Herbivore infestation, wounding and JA treatment enhance expression levels of OsHI-LOX
Northern blot analysis of rice stems and leaves revealed low to undetectable constitutive expression of OSHI-LOX (Figure 1b). While mechanical wounding and BPH feeding led to a slight increase in expression, attack by the SSB caterpillar resulted in an obvious and rapid increase in transcript levels in both infested stems and non-infested leaves, starting <1 h after attack and peaking between 12 and 24 h in the stem after herbivore infestation (Figure 1B). JA treatment induced transcript accumulation of OsHI-LOX, but SA treatment did not (Figure 1B). Hence, OsHI-LOX may be specifically involved in JA-related insect responses in rice.
Silencing OsHI-LOX reduces elicited JA and TrypPI levels, but not GLV levels
OsHI-LOX silencing by Agrobacterium-based transformation (Figure S4) resulted in three independently transformed lines (L102-2, L145-1 and L188-3) with a single insertion and one line (L176-3) with two insertions (Figure S5). Northern hybridization showed that expression of OsHI-LOX in SSB infested stems decreased markedly in three of the as-lox lines (L145-1, L176-3 and L188-3) compared to identically treated WT plants; the expression of OsHI-LOX in line L102-2 decreased only slightly (Figure 2A). We thus chose two as-lox lines (L145-1 and L188-3) with a single insertion and obviously decreased expression of OsH1-LOX for most subsequent experiments. Compared to WT plants, growth of as-lox plants was delayed during their whole development (Figure S6).
JA analysis showed that basal JA levels in the as-lox lines did not differ significantly from those of WT plants except for line L145-1, whose JA levels were significantly reduced (Figure 2b). JA levels in WT plants significantly increased 1.5 and 3 h after infestation with SSB, but those in as-lox plants did not (Figure 2b). Compared to WT plants, the herbivore-induced JA levels in the four as-lox lines were reduced, especially 3 h after the start of herbivore attack (Figure 2b).
We also analyzed the emission of GLVs in as-lox and WT plants. The levels of constitutive and wound-induced emission of (Z)-3-hexenal and (Z)-3-hexen-1-ol from as-lox lines (L145-1 and L188-3) did not differ from those of WT plants (Figure 2c,d). Taken together, these findings suggest that OsHI-LOX supplies substrates for induced JA biosynthesis but not for the production of GLVs in rice.
Constitutive TrypPI levels in as-lox lines did not differ significantly from levels in WT plants except for line L188-3, whose TrypPI levels were significantly reduced (Figure 3a). However, SSB- or BPH-elicited TrypPI activity in as-lox lines L145-1 and L188-3, measured 3 days after the start of herbivore infestation, was significantly reduced compared to levels in herbivore-induced WT plants (Figure 3a,b). Treatment with JA elicited higher TrypPI levels in leaves of WT plants than in leaves of as-lox plants (L188-3), although the induced TrypPI levels in stems of WT and as-lox plants were not statistically different (Figure 3c). Treatment with JA and LF also induced higher TrypPI levels in WT plants than in as-lox plants (Figure 3d). The results demonstrate that JA pathway plays an important, but not exclusive role, in the herbivore-induced production of TrypPI in rice.
Silencing OsHI-LOX reduces resistance against SSB and LF, but enhances it against BPH
SSB caterpillars gained more mass on as-lox lines L145-1 and 188-3 than on WT plants (Figure 4a). By day 7, the masses of caterpillars that fed on L145-1 and L188-3 were 146.1 and 131.3%, respectively, of those that fed on WT plants. A similar result was observed for the chewing herbivore LF. By day 6, the masses of LF caterpillars that fed on as-lox lines L145-1 and L188-3 were 1.51 and 1.31 times higher than the masses of those that fed on WT plants (Figure 4c). Furthermore, JA treatment reduced the increase in mass for LF larvae that fed on both as-lox line L188-3 and WT plants, but the mass increases in LF larvae that fed on JA-treated as-lox plants were still higher than those in larva that fed on JA-treated WT plants, suggesting that as-lox plant resistance was only partially restored by JA elicitation (Figure 4d). Consistent with these findings, as-lox plants were more severely damaged by SSB (Figure 4b) and LF (Figure S7) and fewer survived (Figure 4b).
We also assessed the performance of BPH on as-lox lines. When the various rice genotypes were exposed to a BPH colony, BPH female adults were more often found on WT plants than on as-lox plants. This apparent preference was most significant for as-lox line L145-1 during the 8 h period following the start of the experiment (Figure 5a,b). Similarly, BPH female adults laid significantly more eggs on WT plants than on either as-lox line; the numbers of eggs on the as-lox lines were only 55.6 and 33.8% of the number laid on WT plants, respectively (Figure 5, insets). The amounts of honeydew secreted per day by a BPH female adult, an indicator of the amount of food intake, were reduced by 16.9 and 42.9% on as-lox lines, L145-1 and L188-3, respectively, compared to WT plants (Figure 5c). Similarly, BPH nymphs that fed on the as-lox lines had lower survival rates than those that fed on WT plants, especially nymphs that fed on as-lox line L188-3 (Figure 5d). There were obvious differences between the as-lox lines (L145-1 and L188-3) and WT plants in terms of how they tolerated BPH infestation: WT plants died more rapidly than as-lox plants when they were infested by BPH female adults (Figure 6d). Sixteen days after infestation by 12 BPH female adults, WT plants had completely wilted, whereas only the outer leaf sheaths showed necrosis in as-lox plants (Figure 6b).
Silencing OsHI-LOX results in BPH-induced cell death and increases in SA and H2O2 levels
Although as-lox plants were tolerant of BPH infestation (Figure 6b), the infested local leaf sheaths of as-lox plants became brown and died more quickly than those of WT plants (Figure 6a). Three days after attack by BPH female adults, the damaged sites on leaf sheaths of L188-3 turned into brown spots, and 7 days later, the spots connected to form a large area (Figure 6a). In contrast, even 7 days after attack by BPH, the damaged sites on leaf sheaths of WT plants had only become small light brown spots (Figure 6a). This obvious similarity to a pathogen-induced plant hypersensitive response (HR) suggests that infestation by BPH resulted in HR-like cell death in as-lox plants. Therefore, we determined the concentrations of JA, SA and H2O2 in L188-3 and WT plants at various times after they were infested by BPH female adults. BPH infestation increased JA levels in WT plants at 8 and 48 h (Figure 7b). Consistent with the weak elicitation of JA by BPH, silencing OsHI-LOX did not significantly decrease JA levels in as-lox line L188-3 infested with BPH compared to WT plants (F1,68 = 0.017, P = 0.8963) (Figure 7b); however, silencing OsHI-LOX did inhibit the increases in BPH-induced JA levels at 8 and 48 h after BPH infestation.
SA levels in rice plants infested with BPH were higher compared to SA levels in non-infested plants (F1,77 = 4.85, P = 0.0307). However, this influence was dependent on line (F1,77 = 13.84, P = 0.0004) and treatment time (F6,77 = 11.15, P = 0.0000). In BPH-infested WT plants, SA levels were significantly higher at 3 and 12 h after treatment than they were in the control plants, whereas in BPH-infested plants of as-lox line L188-3, SA levels were significantly higher at 12 and 72 h than they were in the control plants (Figure 7a). Compared to WT plants, as-lox line L188-3 plants had higher SA levels (F1,103 = 5.69, P = 0.0188), especially at 8, 48 and 72 h after treatment (Figure 7a).
There were significantly higher constitutive and induced H2O2 levels in as-lox line L145-1 than in WT plants (F1,51 = 58.90, P = 0.0000; Figure 7c). BPH infestation enhanced H2O2 levels in plants (F1,51 = 12.52, P = 0.0009), depending on line (F1,51 = 4.79, P = 0.0333) and time (F4,51 = 4.37, P = 0.0041). In as-lox line L188-3, H2O2 levels in BPH-infested plants were only significantly higher than H2O2 levels in control plants at 8 and 24 h after treatment; for WT plants, no difference in H2O2 levels was observed between the BPH-infested plants and the control plants (Figure 7c).
We cloned an herbivore-induced LOX gene in rice, which was designated as OsHI-LOX. OsHI-LOX is allelic to OsRLL, a 13-LOX that has a putative chloroplast-targeting transit peptide and is transcriptionally up-regulated upon contact with an incompatible strain of rice blast fungus (Peng et al., 1994). Here, we confirm that OsHI-LOX is localized in chloroplasts and that its transcripts are up-regulated by treatment with SSB caterpillars, BPH, wounding and JA, but not by treatment with SA (Figure 1). Antisense expression of OsHI-LOX decreased herbivore-induced JA levels in rice (Figure 2). These results suggest that OsHI-LOX plays an important role in induced JA biosynthesis. To date, of the five rice LOX cDNAs that have been cloned and characterized, only OsLOX1, a 9/13-LOX, has been implicated in insect-induced JA accumulation (Wang et al., 2008b). Therefore, OsHI-LOX is a type 2 13-LOX in rice that has been identified as being involved in herbivore-induced JA biosynthesis.
By showing that antisense expression of OsHI-LOX has no effect on wound-induced release of GLVs, but decreased herbivore-elicited JA levels (Figure 2), we demonstrate that, in rice, as reported in other plant species such as Solanum tuberosum (potato) (León et al., 2002) and Nicotiana attenuata (Halitschke and Baldwin, 2003), the substrates for the allene oxide synthase (AOS) and hydroperoxide lyase (HPL) pathways are delivered by distinct LOX isoforms, and OsHI-LOX supplies substrates to the AOS pathway but not the HPL pathway.
Silencing OsHI-LOX in rice decreased plant tolerance and improved the larval performance of the two lepidopteran chewing herbivores, SSB and LF (Figures 4 and S7). This coincided with impaired herbivore-induced TrypPI levels (Figure 3), suggesting that the OsHI-LOX-mediated signal cascade plays a central role in rice defense responses, including TrypPI production, induced by the two herbivores. Indeed, TrypPI has been implicated in plant defense against lepidopteran herbivores (Zavala et al., 2004). Thus, the reduction of herbivore resistance in as-lox rice plants can at least partially be explained by a decrease in induced TrypPI activity. The deficiency in herbivore resistance to lepidopteran herbivores (Figure 4) and induced TrypPI accumulation (Figure 3c,d) in as-lox plants could not be fully restored by exogenous application of JA. It is apparent that other herbivore-induced signals, in addition to OsHI-LOX-derived JA, are involved in OsHI-LOX-dependent rice resistance and TrypPI activation. These additional signals could be SA, ethylene or H2O2, all of which were induced by SSB or LF caterpillar feeding (Wang, 2006) and have been reported to be involved in plant defense responses (Orozco-Cárdenas et al., 2001; Bostock, 2005; Koornneef and Pieterse, 2008), or intermediates in the octadecanoid pathway upstream of JA, such as 12-oxo-phytodienic acid (OPDA), which has been shown to up-regulate the expression levels of many defense-related genes and induce the production of numerous secondary metabolites (Blée, 2002).
We also determined the influence of as-lox plants on the performance of BPH, a homopteran phloem feeder of rice. Interestingly, female BPH adults showed a preference for settling, ovipositing and feeding on WT plants rather than on as-lox plants (Figure 5a–c), and BPH nymphs suffered higher mortality on as-lox plants than on WT plants (Figure 5d). These results are consistent with our previous findings that female BPH adults showed a preference for settling on JA-treated plants rather than on control plants (Lou et al., 2005). Moreover, WT plants died more rapidly than as-lox plants when infested with an equal number of BPH nymphs or female adults (Figure 6b). This observation implies an increase in rice resistance to BPH in as-lox plants, in contrast to their response to the two rice lepidopteran herbivores, SSB and LF.
The antagonistic roles played by the JA pathway in affecting plant resistance to chewing and phloem-feeding herbivores in monocotyledonous plants reported here question the general assumption that JA-dependent defenses enhance plant resistance against both chewing and phloem-feeding herbivores. Possible factors that enhance resistance to BPH in as-lox plants could be increased BPH-induced levels of SA and/or H2O2 (Figure 7), BPH-induced HR-like cell death (Figure 6a) or a combination of these factors. For example, silencing the germin-like gene GLP that is involved in H2O2 production in Nicotiana attenuata improves Manduca sexta larval performance (Lou and Baldwin, 2006). Treating tomato plants with acibenzolar in both field and laboratory experiments reduced leaf miner larval densities (Liriomyza spp.) (Inbar et al., 1998). The SA signaling pathway was also found to play an important role in plant resistance to phloem-feeding herbivores (Bostock, 2005; Kaloshian and Walling, 2005; Li et al., 2006), but it is generally assumed that induction of SA is in fact a decoy strategy by phloem feeders to reduce the activation of JA-dependent defenses (Walling, 2008). Our results support the idea that the relative roles of JA and SA in insect resistance can differ substantially depending on the plant species (Goggin, 2007).
Many studies have reported that HR plays an important role in plant defense against pathogens (Mur et al., 2008). The influence of HR or premature senescence on the performance of phloem-feeding herbivores has also been observed in some plants. For example, the premature leaflet senescence of Pistacia palaestina trees induced by a gall aphid reduced the performance of another aphid feeding on the same leaflet (Inbar et al., 1995). In barley, resistance to the Russian wheat aphid was correlated with the activation of cell death in resistant cultivars (Belefant-Miller et al., 1994). Pegadaraju et al. (2005) found that Arabidopsis leaf senescence, modulated by an SA-regulated gene, PHYTOALEXIN DEFICIENT4, was accompanied by enhanced resistance to the green peach aphid Myzus persicae. Here we found that infestation by BPH, especially BPH female adults, induced HR-like cell death on outer leaf sheaths of as-lox plants (Figure 6a), causing the outer leaf sheaths of as-lox plants to die earlier than those of WT plants. Nutrient export from the senescing leaf sheaths (Pegadaraju et al., 2005) could have reduced BPH feeding. Moreover, death of the outer leaf sheath, which acts as a protective layer, could deter BPH from feeding and oviposition. Determining the plant responses to BPH infestation will elucidate the mechanisms by which as-lox plants resist BPH.
Activation of HR-like cell death in as-lox plants was stronger in response to infestation by BPH female adults than by BPH nymphs, suggesting that the HR elicitors mainly derive from BPH eggs and/or ovipositor exudates. Analysis of the JA, SA and H2O2 levels showed that, unlike WT plants, BPH infestation of as-lox plants did not increase JA levels and was accompanied by higher constitutive and induced SA and H2O2 levels (Figure 7). We previously reported that infestation by BPH female adults does not induce increases in JA levels in plants of the susceptible rice variety TN1 (Wang et al., 2008c). Using a microarray with 108 cDNAs, Zhang et al. (2004) also found that none of the genes involved in JA biosynthesis were up-regulated by BPH nymph infestation of either resistant (B5) or susceptible (MH63) rice varieties. In contrast, Wang et al. (2008b) reported that BPH infestation induced JA biosynthesis in the rice variety Beilu PL 2. Their results and ours demonstrate that BPH activation of the JA pathway in rice plants differs between genotypes. SA, and in particular H2O2, have been shown to play central roles in HR (Mur et al., 2008). In a rice line expressing a fungal glucose oxidase gene, elevated endogenous levels of H2O2 led to cell death (Kachroo et al., 2003). Moreover, in Arabidopsis, JA-insensitive and -deficient mutants display increased levels of O3-induced active oxygen species as well as SA accumulation and a heightened HR to ozone (Rao et al., 2000). Therefore, the stronger BPH-induced HR in as-lox plants compared to WT plants is most likely related to higher induced SA and H2O2 levels, which in turn are probably due to lack of JA induction after silencing of OsHI-LOX (Figure 7b). This demonstrates an important modulating role of the OsHI-LOX-mediated pathway in the BPH-induced HR. In contrast to dicotyledonous plant–insect interaction models, the absence of JA-dependent defenses and the induction of an SA response appear to increase resistance against phloem feeders in rice. In this case, an SA-dependent response upon attack by BPH is advantageous for the plant rather than a manipulative decoy by the herbivore. Although it may be too early to speculate about the possibility of BPH requiring an intact JA pathway in order to be able to reduce SA- and H2O2-mediated defenses, our results clearly warrant revision of current models of monocotyledonous plant responses to chewing and phloem-feeding insects.
The rice genotypes used in this study were Xiushui 11 (wild-type, WT) and as-lox transgenic lines (see below). Pre-germinated seeds were sown in a greenhouse, and, after 20–25 days, seedlings were transplanted to small clay pots (diameter 8 cm, height 10 cm) each containing one plant, or large clay pots (diameter 16 cm, height 14 cm) each with two plants (one WT plant and one as-lox transgenic line plant). Plants were watered daily, and each pot was supplied with 10 ml of nutrient solution (urea, 1 g l−1) every week. All plants were grown in a greenhouse at 28 ± 2°C, with a 12 h light phase and 70–80% relative humidity. Plants were used for experiments 25–30 days after transplanting.
Colonies of SSB, LF and BPH were originally obtained from rice fields in Hangzhou, China, and maintained on Xiushui 11 rice seedlings in a controlled climate room at 26 ± 2°C, 12 h light phase and 80% relative humidity.
Generation and characterization of as-lox transgenic lines
For the plant transformation vector, a 763 bp portion of the OsHI-LOX cDNA present on plasmid pLOX (see below) was PCR-amplified using primers 5′-CATCTGGGACGCCATCAAG-3′ and 5′-ATAAAGATTTGGGAGTGACATA-3′. The obtained 0.76 kb PCR fragment was cut using BamHI and SalI. The resulting 0.76 kb fragment (Figure S1) was cloned into pCAMBIA1301, yielding the transformation vector pCAMBIA-LOX (13.4 kb) (Figure S4) containing the hygromycin resistance gene hph and GUS reporter gene as selectable markers. The OsHI-LOX gene fragment was present in an antisense orientation downstream of the 35S promoter and upstream of the terminator on the T-DNA of the resulting binary plant transformation vector, thus enabling transcription of OsHI-LOX antisense RNA. This vector was used for transforming rice variety Xiushui 11 using an Agrobacterium-mediated transformation procedure (Hiei et al., 1994). The progeny of homozygous plants were selected by hygromycin resistance screening or GUS staining. The number of insertions was determined by Southern hybridization of genomic DNA using a PCR fragment of the GUS gene as a probe. For most experiments, two T2 homozygous lines, L145-1 and L188-3, each harboring a single insertion (Figure S5), and WT plants were used.
SSB treatment. Pots with one plant were used for this experiment. Plants were individually infested using a third-instar larva of SSB that had been starved for 2 h. Control plants (C) were not manipulated.
LF treatment. Plants (one per pot) 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). Control plants (C) were not manipulated.
BPH treatment. Plants (one per pot) were individually infested with 16 gravid BPH females contained in two parafilm bags (6 × 5 cm, with 60 small holes made by a needle, each bag containing eight females) fixed to upper and lower positions on the plant stems. Two empty parafilm bags were attached to control plants (non-infested).
Mechanical wounding. Plants (one per pot) were individually damaged using a needle on the lower part of rice stems (about 2 cm long), each with 200 pricks (group W). Control plants (C) were not manipulated.
JA and SA treatment. Pots with one plant were used for the experiments. Previous experiments had found that exogenous application of JA (16.7–167 μg/plant) and SA (100–200 μg/plant) was sufficient to elicit volatile production (Lou et al., 2005) and TrypPIs (Wang, 2009), respectively. Thus, in this experiment, plants were individually sprayed with 2 ml of JA (100 μg ml−1) or SA (70 μg ml−1) in 50 mm sodium phosphate buffer (titrated with 1 m citric acid to pH 8, with 0.01% Tween). Control plants were sprayed with 2 ml of the butter (BUF group).
Isolation of the full-length cDNA of OsHI-LOX
OsHI-LOX cDNA fragments were obtained by RT-PCR from total RNA isolated from WT plants infested by an SSB instar for 24 h. The primers OsHI-LOX-F (5′-ATAATACCCATACCATGTTGCG-3′) and OsHI-LOX-R (5′-ATAAAGATTTGGGAGTGACATA-3′) were designed based on the sequence of rice lipoxygenase gene OsRLL (accession number D14000), which has a high homology with the partial sequence of OsHI-LOX that was cloned by suppression subtractive hybridization (SSH) and found to be elicited by SSR larvae feeding (G. Z. and Y.L., unpublished results). The PCR product was cloned into pGEM-T Easy vector (Promega, http://www.promega.com/) (pLOX) and sequenced. Amino acid sequences of LOX-related proteins were aligned using MegAlign (DNASTAR, http://www.dnastar.com). Phylogenetic relationships were determined using the neighbor-joining method.
Northern blot analysis
Samples were ground in liquid nitrogen and total RNA was extracted using Trizol (Invitrogen, http://www.promega.com/) according to the manufacturer’s instructions. Total RNA samples (20 μg) were size-fractionated by 1.2% w/v agarose formaldehyde gel electrophoresis, and capillary blotted onto a Hybond N+ nylon membrane (Millipore, http://www.millipore.com) as described in the manufacturer’s instructions. Ethidium bromide staining of the gel prior to blotting revealed rRNA bands that served as a loading control. After blotting and UV cross-linking, α-32P-labeled probes specific for OsHI-LOX were used for detection. The probe was obtained by PCR based on the partial cDNA sequence of OsHI-LOX (Figure S1) with random primers using the manufacturer’s protocol for the TaKaRa random primer labeling kit (TakaRa, http://www.takara-bio.com). Hybridization conditions were as follows: pre-hybridization at 62°C with hybridization buffer (0.25 m NaHPO4, pH 7.2, 7% SDS, 1 mm EDTA, 1% BSA) for 2 h, hybridization at 62°C for 14 h, and washing with 2 × SSC and 0.1% SDS (25°C) twice each for 20 min, followed by 0.1 × SSC and 0.1% SDS (62°C) once for 10 min. After autoradiography on a storage phosphor screen (Amersham Biosciences, http://www5.amershambiosciences.com/), imaging was performed using a Typhoon 8600 scanner (Amersham Biosciences).
Southern blot analysis
Genomic DNA was extracted from rice leaves using a cetyl trimethyl ammonium bromide procedure (Ausubel et al., 1987). DNA (20 μg) was digested using various restriction enzymes, size-fractionated in a 0.8% w/v agarose gel, and transferred to a nylon membrane (Hybond N+, Millipore). To determine the number of insertions in transgenic plants, the sequence of the GUS reporter gene was used as a probe for Southern hybridization. The probe was labeled with α-32P (random primer labeling kit, TaKaRa). Hybridization conditions and imaging were the same as for the RNA gel-blot analysis.
OsHI-LOX mRNA expression analysis
To analyze the effects of various stresses on expression levels of OsHI-LOX mRNA, WT plants were randomly assigned to one of eight treatments: JA, SA, BUF, W, SSB, BPH, non-infested and C. The leaves at node 4, stems or both were harvested at various times after the start of treatment (see Figure 1). Total RNA was extracted for all samples, after which OsHI-LOX mRNA expression levels were detected by Northern blot.
To examine the efficiency of antisense suppression, OsHI-LOX mRNA expression levels in stems of the four as-lox lines (L102-2, L145-1, L176-3 and L188-3) and WT plants that were individually infested by an SSB instar for 24 h were measured by Northern blot.
Subcellular localization of OsHI-LOX
The OsHI-LOX cDNA full-length ORF without the stop codon was fused to GFP (accession number U87973). The fusion gene was inserted into pCAMBIA1301, yielding the transformation vector pLOX-GFP (Figure S8). This vector was used for transient transformation of Nicotiana tabacum leaves as described by Wroblewski et al. (2005). The epidermal cells of N. tabacum leaves were observed for subcellular localization of OsHI-LOX using a confocal laser scanning microscope (Leica TCS SP5-DMI6000, http://www.leica.com). GFP fluorescence was excited at 488 nm and detected using a 500–530 nm emission filter; chlorophyll autofluorescence was excited at 543 nm and detected using a 620–700 nm emission filter.
SA, JA and GLV analysis
Plants (one plant per pot) were randomly assigned to SSB, C, BPH and non-infested treatment. For SSB and C treatment, four as-lox lines (L102-2, L145-1, L176-3 and L188-3) and one WT line were used, and the stems were harvested at 0, 1.5 and 3 h after treatment, the time of obvious JA accumulation after SSB infestation (Wang, 2006). For BPH and non-infested treatment, one as-lox line L188-3 and one WT line were used, and the leaf sheaths were harvested at 0, 3, 8, 12, 24, 48 and 72 h after treatment. Samples were immediately immersed in liquid nitrogen and stored at −80°C. For each treatment and each time interval, five plants were sampled. JA and SA were extracted for GC-MS analysis using labeled internal standards (328 ng D3-JA, kindly supplied by Ian T. Baldwin, Max Planck Institute of Chemical Ecology, Jena, Germany, and 345 ng D6-SA, Cambridge Isotope Laboratory, Cambridge, MA, USA) as described by Lou and Baldwin (2003).
GLV emissions [(Z)-3-hexenal and (Z)-3-hexen-1-ol] were analyzed using a method similar to that described by Halitschke and Baldwin (2003). GLVs were sampled in a 50 ml glass chamber for 40 sec from part of individual leaves (0.02 g) before and immediately after leaves were cut into small pieces (2 mm long), and analyzed using a portable gas analyzer (zNose™ 4200, Electronic Sensor Technology, http://www.estcal.com). Five to 10 plants were used for each genotype (L145-1, L188-3 and one WT line). (Z)-3-hexenal and (Z)-3-hexen-1-ol concentrations were expressed as peak area per g of fresh leaves.
Quantification of hydrogen peroxide
WT plants and plants of as-lox line L188-3 were randomly assigned to BPH and non-infested treatment. Leaf sheaths were harvested at 0, 3, 8, 12 and 24 h after treatment. For each treatment and each time interval, five plants were sampled. The homogenized samples were individually mixed with 1 ml of deionized water, and the supernatants were collected by microcentrifugation (13 600 g) of the extract at 4°C for 10 min. H2O2 concentrations were then determined as described by Lou and Baldwin (2006).
Plants (one plant per pot) from each line (L145-1, L188-3 and one WT line) were randomly assigned to SSB, BPH, non-infested, JA, BUF, JA + LF, BUF + LF and C groups. For JA + LF and BUF + LF treatment, the plants were treated for 1 day with JA or the buffer as described above, followed by infestation with LF third-instar larvae on leaves at node 4 (one larva per plant). Treated leaves or stems (0.2–0.3 g per sample) were harvested 3 days (SSB, BPH, non-infested, JA, BUF and C treatment) or 5 days (BUF + LF and JA + LF treatment) after the start of herbivore infestation. The samples were immediately immersed in liquid nitrogen and stored at −80°C. The TrypPI concentration was measured using a radial diffusion assay as described by van Dam et al. (2001). Each treatment at each time interval was replicated six times.
SSB and LF performance measurement. Freshly hatched SSB larvae were allowed to infest stems of plants of as-lox lines L145-1 and L188-3 and WT plants (five larvae per plant). Ten to 15 plants were used for each line. Larval mass (to an accuracy of 0.1 mg) was measured 7 days after the start of the experiment.
For LF performance measurement, second-instar larvae, which had been weighed and starved for 2 h, were placed individually on the node 4 leaf of plants of as-lox lines L145-1 and L188-3 and one WT line. In complementation experiments, transgenic line L188-3 and WT plants were used, and larvae were placed individually on plants that were either treated with JA, the buffer (BUF) or not manipulated (C) for 1 day. Twenty-five to 30 plants were used for each line. Larval mass was measured to an accuracy of 0.1 mg 5 days after the larvae were placed on plants, and the increased percentages of larval mass on each line were calculated.
BPH performance measurement. To determine the colonization and oviposition preferences of BPH, pots with two plants (an as-lox line plant and a WT plant) were individually confined within plastic cages (diameter 12 cm, height 36 cm) into which 12 gravid adult BPH females were introduced. The number of BPH on each plant was calculated at 1, 2, 4, 8, 24 and 48 h after the release of BPH, and then, 72 h later, BPH were removed and the eggs on each plant were counted under a microscope. The experiment was repeated 6–8 times.
BPH feeding was measured on WT and as-lox transgenic lines. A newly emerging macropterous female BPH adult was placed into a small parafilm bag (6 × 5 cm), which was then fixed on the stems of plants, with each plant receiving two females. The amount of honeydew excreted by a female adult was weighed (to an accuracy of 0.1 mg) 24 h after the start of the experiment. Two as-lox lines and one WT line were used for this experiment. The experiment was replicated 20 times.
The survival rates of BPH nymphs on WT and as-lox plants were also determined. Pots with one plant were individually covered with plastic cages (diameter 9 cm, height 36 cm) into which 15 newly hatched BPH nymphs were released. The number of surviving BPH nymphs on each plant was recorded every day until 12 days after the introduction of the herbivore. The experiment was repeated 8–10 times.
Plant tolerance measurement. To determine the differences in plant tolerance to herbivore attack between WT and as-lox plants, plants of as-lox lines L145-1 and L188-3 and one WT line were individually infested by a SSB third-instar larva or 12 gravid BPH female adults that had been confined in plastic cages (diameter 9 cm, height 36 cm). Every day, the damage levels of plants were checked and photographs were taken, and the number of BPHs was checked and dead insects were replaced.
Differences in herbivore performances the various lines and TrypPI levels in JA, BUF and C plants were determined using Student’s t-tests Differences in SSB-induced JA levels, herbivore-elicited TrypPI levels, increased LF mass on JA, BUF and C plants, and GLVs were analyzed by one-way anova; if the anova analysis was significant (P < 0.05), Duncan’s multiple range test was used to detect significant differences between groups. Differences in BPH-induced JA, SA and H2O2 levels were analyzed by three-way anova with genotype (WT and L188-3), treatment (BPH, non-infested) and time as main effects, plus all interaction terms. Means were separated using Duncan’s multiple range test. The mean separations shown in Figure 7 reflect differences between treatments at each time only. Data were analyzed using Statistica (SAS Institute Inc., http://www.statsoft.com).
We thank Xia Wang and Jing Lv for their invaluable assistance with laboratory work, and Emily Wheeler (Department of Molecular Biology, Max Planck Institute of Chemical Ecology, Jena) for invaluable editorial assistance. We thank Ted Turlings (Institute of Biology, University of Neuchâtel) for his help in revising an earlier version. The study was jointly sponsored by the National Basic Research Program of China (2006CB102005), the Division of Science and Technology of Zhejiang Province (2006C22003), the Natural Science Foundation of Zhejiang Province (D3080282) and the Innovation Research Team Program of the Ministry of Education of China (IRT0535).
The GenBank accession number for OsHI-LOX is bankit1171099.