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(E)-β-caryophyllene functions as a host location signal for the rice white-backed planthopper Sogatella furcifera
Physiological and Molecular Plant Pathology 91 (2015) 106e112
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(E)-b-caryophyllene functions as a host location signal for the rice white-backed planthopper Sogatella furcifera Qi Wang a, b, Zhaojun Xin a, c, Jiancai Li a, Lingfei Hu a, Yonggen Lou a, Jing Lu a, * a
State Key Laboratory of Rice Biology, Institute of Insect Science, Zhejiang University, Hangzhou 310058, China State Key Lab of Biocontrol, School of Life Sciences, Institute of Entomology, Sun Yat-sen University, Guangzhou 510275, China c Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 December 2014 Received in revised form 24 June 2015 Accepted 4 July 2015 Available online 7 July 2015
Terpenoids are well known to mediate interactions among plants, herbivores and their natural enemies. In rice, (E)-b-caryophyllene has been reported to attract a major insect pest, Nilaparvata lugens, and its egg parasitoid, Anagrus nilaparvatae. Yet, it remains unclear whether (E)-b-caryophyllene also influences other herbivores in rice. Here we demonstrated that a rice (E)-b-caryophyllene synthase, OsCAS, localizes in cytoplasm and that its transcript was up-regulated following mechanical wounding, the infestation of the rice striped stem borer (SSB) Chilo suppressalis and treatment with jasmonic acid. We also revealed that the rice white-backed planthopper Sogatella furcifera preferred feeding and ovipositing on wild-type (WT) plants over feeding on mutants with low levels of (E)-b-caryophyllene. The results indicate that (E)b-caryophyllene may function as an important signal by which herbivores on rice locate their host. © 2015 Hainan Medical University. Published by Elsevier Ltd. All rights reserved.
Keywords: (E)-b-caryophyllene Rice Sogatella furcifera Plant volatile Planteherbivore interaction
1. Introduction Herbivore-induced plant volatile organic compounds (VOCs) play an important role in regulating interactions among plants, herbivores and their natural enemies [1e5]. Terpenoids, the main element of which VOCs are composed, are synthesized via mevalonate (MVA) or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways in both mono- and dicot plants [6e8]. Terpenoids are known to serve as attractants or repellents to herbivorous insects and their parasitoids and predators [9e12]. Nicotiana attenuata, for example, when attacked by herbivores, releases a considerable amount of cis-a-bergamotene and then recruits carnivores Geocoris pallens and Geocoris punctipes to counter-attack the pest Manduca sexta [13,14]. Similarly, (E)-b-farnesene emitted from Arabidopsis attracts the parasitoid Diaeretiella rapae but has a repellent effect on the aphid Myzus persicae [15]. In addition, terpenoids can be used directly by plants for priming their own defenses before herbivore attack [16e18]. For example, the exogenous application of b-ocimene on Arabidopsis increased the expression of the genes which
* Corresponding author. E-mail addresses: [email protected] (Q. Wang), [email protected] (Z. Xin), [email protected] (J. Li), [email protected] (L. Hu), yglou@zju. edu.cn (Y. Lou), [email protected] (J. Lu).
are responsible for the octadecanoid pathway [19]. These results indicate the complex ecological roles of terpenoids in modulating tritrophic interactions. (E)-b-caryophyllene, a sesquiterpene, has been reported to play an important role in mediating tritrophic interactions. In 2005, for example, Rasmann found that (E)-b-caryophyllene emitted from maize roots strongly attracts an entomopathogenic nematode of the pest Diabrotica virgifera virgifera. Likewise, elevated (E)-b-caryophyllene from leaves infested by Spodoptera litura strongly attracts Cotesia marginiventris, a larval parasitoid of the herbivore [20]. In addition, a recent study also showed that (E)-b-caryophyllene and ethylene were used by D. virgifera virgifera larvae to locate the maize plants which are the most suitable for their development [21]. However, very little is known about how (E)-bcaryophyllene functions in other plant species. Rice, one of the most important food crops in the world, suffers heavily from insect pests, such as piercing-sucking herbivores, brown planthopper (BPH) Nilaparvata lugens and white-backed planthopper (WBPH) Sogatella furcifera, and chewing herbivores, striped stem borer (SSB) Chilo suppressalis and leaf folder Cnaphalocrocis medinalis [22]. Previous studies revealed that herbivore attack induces a set of defensive response in rice by activating the signaling pathways (e.g. jasmonate and ethylene signaling), by expressing defensive genes and by producing both direct (e.g.
http://dx.doi.org/10.1016/j.pmpp.2015.07.002 0885-5765/© 2015 Hainan Medical University. Published by Elsevier Ltd. All rights reserved.
Q. Wang et al. / Physiological and Molecular Plant Pathology 91 (2015) 106e112
trypsin protease inhibitors) and indirect (e.g. VOCs) defense compounds [23e26]. In rice, (E)-b-caryophyllene could be induced by SSB or fall armyworm infestation and treatment with jasmonic acid (JA) but not by BPH infestation [25,27e29]. Using transformed rice plants overexpressing an (E)-b-caryophyllene synthase gene, OsCAS (namely OsTPS3), Cheng et al. showed that (E)-b-caryophyllene attracts Anagrus nilaparvatae [30], an egg parasitoid of rice planthoppers. Moreover, Fujii et al. found that (E)-b-caryophyllene emitted from flowering rice panicles attracts the rice leaf bug Trigonotylus caelestialium [31]. Recently, our study revealed that the constitutively produced (E)-b-caryophyllene in rice is attractive to BPH as well as to its parasitoids and predators [25]. It seems that (E)-b-caryophyllene plays an important role in mediating interactions among rice plants, herbivores and their natural enemies. However, little is known about the effect of (E)-b-caryophyllene on other insects on rice except for the effect on BPH and their natural enemies. In this study, we chose WBPH, a major pest of rice which severely damages rice yield by sucking phloem sap and transmitting the southern rice black-streaked dwarf virus [22,32], as a testing insect and carried out experiments using ir-cas transformed rice plants with silenced expression of OsCAS by the RNAi technology [25]. We asked the following questions: 1) Does (E)-b-caryophyllene influence the host-searching and oviposition behavior of WBPH in the lab? 2) Does (E)-b-caryophyllene affect the population dynamics of WBPH in the field? Our results provide new evidence for the importance of (E)-b-caryophyllene in riceeherbivore interactions.
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third-instar SSB larva that had been starved for 2 h. Nonmanipulated plants were used as controls (C). For the WBPH treatment, plants were individually infested with 15 gravid female adults that were confined in a glass cage (4-cm diameter, 8-cm height, with 48 0.8-mm diameter holes). Control plants were placed in a similar cage without herbivores (Non-infested). For JA and salicylic acid (SA) treatments, plants were individually sprayed with 2 ml of JA (100 mg ml1) or SA (70 mg ml1) in 50 mM sodium phosphate buffer. Control plants were sprayed with 2 ml of the buffer (BUF). 2.4. Subcellular localization of OsCAS A 210-bp sequence was amplified by PCR from the 50 -end of the OsCAS ORF using primers OsCASsp-FP (50 -ATCCATGGCAACCTCTGTTCCGAGTGT-30 ) and OsCASsp-RP (50 -ATCCATGGATTCCTCGGAAACATGAGCT-30 ) (bold letters represent the NcoI digest site). This fragment was inserted into the pEGFP vector (BD Biosciences, San Jose, CA, USA) to fuse it with EGFP. The fusion gene OsCAS::EGFP was then inserted into the transformation vector pCAMBIA1301, yielding pCAMBIA1301-OsCAS::EGFP (Fig. S2A). Meanwhile, the full-length open reading frame (ORF) of EGFP was inserted into pCAMBIA1301, yielding pCAMBIA1301-EGFP (Fig. S2B). These two vectors were used separately to transiently transform leaves of Nicotiana tabacum as described by Zhou et al. [26]. Fluorescence analysis was performed as described by Zhou et al. [26]. 2.5. Gus staining and quantitative GUS activity assay
2. Materials and methods 2.1. Plants and insects Rice genotypes used in this study were wild-type (WT) Xiushui11 (XS11), two ir-cas transgenic lines, L-cas45 and L-cas51 [25] and an OsCASp::GUS reporter line L41 (see below). Generally, hydroponic plants of lines XS11, L41, L-cas45 and L-cas51 with an age of 45e50 days were used for experiments as described by Wang et al. [33]. Some L41 plants were cultivated in soil until earing for organ-specific GUS staining. Colonies of WBPH and SSB were originally obtained from a rice field in Hangzhou, China, and were maintained on Xianyou 63 seedlings. 2.2. Generation and characterization of the OsCASp::GUS reporter line A 2.5-kb genomic DNA region of OsCAS (NCBI: DQ872158), including promoter and transcription initiation regions, was PCRamplified by using primers OsCASp-FP (50 -GCGTCGA0 CATCTGAGTAACAACTTGGGTAA-3 ) and OsCASp-RP (50 -CGAATTCTGAGACTAGCATATCGGTGC-30 ) (bold letters represent digest sites of enzymes SalI and EcoRI, respectively). The amplified fragment was then inserted into the transformation vector pCAMBIA-1391 to fuse it with GUS. Transforming rice, screening homozygous T2 plants and identifying the number of insertions was carried out following Zhou et al. [26]. The homozygous line L41 harboring a single insertion of OsCASp::GUS (Fig. S2) was used for subsequent experiments. 2.3. Plant treatments For the mechanical wounding treatment, rice stems (about 2-cm long of the stems' lower part) were individually damaged using a needle 200 times (W). Control plants (C) were not pierced. For the SSB treatment, plants were individually infested with a single
Plant organs were observed under a microscope after immersion in GUS staining buffer (0.1 M sodium phosphate buffer pH 7.0, 10 mM EDTA, 1 mM X-Gluc and 0.01% Triton X-100) at 37 C for 2e8 h. A quantitative GUS activity assay was performed as described by Xin et al. [34]. Plant tissues were homogenized in extraction buffer (50 mM potassium phosphate, 10 mM EDTA, 0.1% Sarcosyl and 0.1% Triton X-100) and centrifuged. An aliquot of the supernatant was incubated with 4-methylumbelliferyl-b-D-glucuronide (4-MUG) as substrate at 37 C for 30 min. The amount of 4methylumbelliferone (4-MU) product was measured with a DTX880 Multimode Detector (Beckman, Fullerton, CA, USA); another aliquot of the supernatant was used to determine protein concentration according to the Bradford protein assay. 2.6. Quantitative real-time PCR (qPCR) qPCR analysis was performed as described by Wang et al. [33]. Four independent replicates were carried out on XS11 and ir-cas transgenic lines. The following primers and probes were used for qPCR of OsCAS and OsActin (Os03g50885): OsCAS-FP (50 TCGACGCTACGAGATGCTTTTA-30 ), OsCAS-RP (50 -CACCGTAGCAGCTACCTGATCT-30 ), OsActin-FP (50 -TGGACAGGTTATCACCATTGGT-30 ), OsActin-RP (50 -CCGCAGCTTCCATTCCTATG-30 ), OsCAS-Probe (50 CGAGGTGAAATGGCGCTCTGAAGG-30 ) and OsActin-Probe (50 CGTTTCCGCTGCCCTGAGGTCC-30 ). 2.7. Analysis of volatiles Collection, isolation and identification of rice volatiles were carried out as described by Lou et al. [27]. Volatiles emitted from XS11, L-cas45 and L-cas51 plants that were infested with WBPH for 24 h, or non-infested were collected. Quantities for each compound were expressed as percentages of peak areas relative to the internal standard (IS, diethyl sebacate) per 8 h of trapping for one plant. Five independent collections were replicated for each treatment.
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Fig. 1. Localization of OsCAS in Nicotiana tabacum cells. Leaves of N. tabacum were transformed with EGFP and OsCAS::EGFP. After incubation for 36 h, the transformed cells were observed under a confocal microscope. Photographs were taken in UV light (lane 1), visible light (lane 2) and in combination (overlay, lane 3). Fluorescence of EGFP was excited at 488 nm and detected using a 500e530 nm emission filter (lane 1).
2.8. WBPH preference WBPH colonization and oviposition preference was determined as described by Zhou et al. [26]. Fifteen gravid females or 18 male adults were released into a cylinder containing two plants (one WT plant versus one ir-cas line, Fig. S4). The number of WBPH individuals on each plant was recorded at different time points, and the eggs were counted at 72 h post release. The experiment was replicated nine times. 2.9. Field experiment To evaluate the effects of (E)-b-caryophyllene on the population dynamics of WBPH in the field, we carried out an experiment with XS11 and two ir-cas lines in Changxing, Zhejiang, China, in autumn 2010. A rice field consisting of nine plots (3 m 3 m, 225 hills of plants) was used. Each plot was surrounded by a rice buffer zone of 1 m. Three plots were randomly assigned to each line. In total, each line was represented by 675 plants that were assigned to three independent replicate plots. The number of WBPH nymphs, female and male adults in each block was investigated on 28 August and 6 and 14 September. 2.10. Data analysis Differences in the levels of GUS activity, OsCAS transcripts, volatiles and WBPH preference and population were determined using one-way ANOVA or Student's t-test. If the ANOVA was significant, Duncan's multiple range tests were performed to detect significance among groups. In the case of non-normality and/or unequal variances, data were transformed prior to ANOVA. SPSS 17 (IBM, Armonk, NY, USA) was used for all analyses. 3. Results 3.1. Subcellular localization of OsCAS In this study, we investigated the subcellular localization of
OsCAS using a confocal microscopic assay of tobacco cells that were transiently expressed with an OsCAS::EGFP fusion protein. The fluorescence of OsCAS::EGFP was observed in nuclei, cytoplasm and cell membranes (Fig. 1), suggesting that OsCAS is a classic cytosolic sesquiterpene synthase. 3.2. Expression pattern of OsCAS To better understand the transcriptional characterization of OsCAS, we cloned a 2.5-kb promoter sequence of OsCAS, fused it to GUS reporter gene and constructed OsCASp::GUS transgenic rice plants, and finally obtained a homozygous T2 line: L41 (Fig. S1). Using a staining assay, the GUS activity was observed in callus, roots, seedlings, leaf sheaths, stems, leaves and glumes, especially on the cut edges of the vegetative organs, but no activities were detected in mature seeds and stamens (Fig. 2a). A quantitative assay revealed the highest GUS activity in leaves, followed by in spikelets and stems; the lowest activity was found in roots (Fig. 2b). We also found that mechanical wounding induced a short-term elevation of GUS activity, whereas SSB feeding or exogenous JA application induced a long-lasting elevation (Fig. 2, cee). SA treatment, on the other hand, moderately inhibited GUS activity for 4e8 h (Fig. 2f). In addition, qPCR assay showed that transcripts of OsCAS were significantly up-regulated at 48 and 72 h after WBPH infestation (Fig. 2g). Taken together, these results indicate that OsCAS is a tissue-specific gene that is responsive to herbivory. 3.3. Silencing OsCAS decreases the level of (E)-b-caryophyllene but not other volatiles in plants infested by WBPH As expected, WBPH-induced transcript levels of OsCAS in both ir-cas transgenic lines: L-cas45 and L-cas51 were only 17.14 and 21.17% of levels of WT plants (Fig. 3, insert). We next collected and analyzed the volatiles emitted from WT and ir-cas plants that were attacked by WBPH. No difference was observed between WT plants and ir-cas lines in constitutive levels of volatiles except for (E)-bcaryophyllene, which was significantly lower in ir-cas lines than in WT plants (Fig. S3). After one day of WBPH infestation, WT plants
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Fig. 2. Organic and elicited GUS activity in OsCASp::GUS transgenic rice plants. (a) GUS staining of (1) callus, (2) mature seeds, (3) 1-day-old seedlings, (4) 3-day-old seedlings, (5) sheaths at the 4-leaf phase, (6) stems, (7) sheaths, (8) leaves, (9) spikelets (especially in glumes) and (10) stamens; (b) mean GUS activity (þSE, n ¼ 6) of OsCASp::GUS transgenic plants. Letters indicate significant differences among organs (P < 0.05, one-way ANOVA and Duncan's multiple range test). (c) to (f) Mean GUS activity (þSE, n ¼ 4 to 5) of OsCASp::GUS transgenic rice stems that were (a) mechanically wounded, (b) infested by the striped stem borer (SSB), (c) treated with jasmonic acid (JA) or (d) salicylic acid (SA). C, control; BUF, buffer. (g) Mean expression levels (þSE, n ¼ 4) of OsCAS in stems of rice plants infested with the white-backed planthopper (WBPH). Asterisks indicate significant differences in transcript levels between treated and control plants (*, P < 0.05; **, P < 0.01; Student's t-test).
up-regulated the emission of the most abundant monoterpene Slinalool, as well as several major sesquiterpenes, including (E)-abergamotene, sesquisabinene A, b-bisabolene, b-sesquiphellandrene and (E)-g-bisabolene. In contrast, no differences in the amount of (E)-b-caryophyllene from WBPH-infested and from noninfested WT plants were observed (Fig. 3). Again, both ir-cas lines revealed significantly lower emissions of (E)-b-caryophyllene: compared with (E)-b-caryophyllene emissions from WBPHinfested WT plants, emissions of (E)-b-caryophyllene decreased
by 78.32 and 95.45% in L-cas45 and L-cas51 lines, respectively. However, silencing (E)-b-caryophyllene did not influence other monoterpenes and sesquiterpenes (Fig. 3).
3.4. Silencing OsCAS reduces the preference of WBPH We investigated the preference of feeding and ovipositing behavior of WBPH on WT and ir-cas plants. As shown in Fig. 4a, compared with two ir-cas lines, WT plants were more attractive to
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Fig. 3. Analysis of WBPH-induced rice volatiles and expression levels of OsCAS. Relative amounts (þSE, n ¼ 5) of volatiles emitted from wild-type (WT) and ir-cas transgenic plants for 8 h. Insert: Mean expression levels (þSE, n ¼ 4) of OsCAS in WT (XS11) or ir-cas plants infested with WBPH for 24 h. Asterisks indicate significant differences between noninfested and WBPH-infested WT plants (*, P < 0.05; **, P < 0.01; Student's t-test). Letters indicate significant differences among lines under WBPH infestation for 1 day (P < 0.05, one-way ANOVA and Duncan's multiple-range test).
WBPH female adults as places to colonize and on which to oviposit: the numbers of female adults that fed on WT plants were 1.29- and 1.93-fold the number of those that fed on L-cas45 and L-cas51 lines at the start of release; similarly, the numbers of eggs laid on WT plants were 41.85 and 99.07% higher than those laid on L-cas45 and L-cas51 lines, respectively. It is noteworthy that the preference appeared at the start of release and subsequently declined. Additionally, WBPH male adults showed a similar colonization preference for WT plants (Fig. 4b). 3.5. Silencing OsCAS decreases the population density of WBPH in the field We next assessed effects of (E)-b-caryophyllene on WBPH population in the field. In accordance with the preference assay in the lab, our results showed that the population densities of WBPH female and male adults and nymphs on ir-cas lines were lower than the population densities on WT plants, especially in the peak period of the population (Fig. 5). The number of WBPH female adults on L-cas45 and L-cas51 lines was only 48.28 and 38.51% (28 Aug), 50.29 and 31.58% (6 Sep) and 72.88 and 52.54% (14 Sep) of that on WT plants, respectively. Likewise, the population densities of WBPH nymphs in the two ir-cas blocks were reduced by 30e50% compared with those in WT blocks on 28 Aug and 6 Sep. 4. Discussion In plants, monoterpenes are mainly synthesized via the MEP pathway in plastids, whereas sesquiterpenes are mainly synthesized in cytoplasm through the MVA pathway [35,36]. As expected, we found that OsCAS is a cytosolic sesquiterpene synthase (Fig. 1), indicating that (E)-b-caryophyllene in rice is synthesized in cytoplasm. We measured the expression pattern of OsCAS in different organs in rice and found that OsCAS was expressed in many organs, mainly in leaves, followed by in spikelets and stems, with the lowest activity found in roots (Fig. 2). This result was similar to that reported in Cheng et al. [30]. Fujii et al. revealed that during the flowering stage, rice panicles released (E)-b-caryophyllene [31]. We
detected GUS activity in glumes but not stamens, implying that glumes might be the main organ for producing (E)-bcaryophyllene. The transcript level of OsCAS could be induced by wounding and by infestation by SSB or WBPH (Fig. 2c, d and g). However, the response of OsCAS to these treatments was different: fast and shortlived for mechanical wounding, fast and long-lived for SSB infestation, and slow and long-lived for WBPH. This discrepancy corresponded to the result of volatiles released from rice plants. For instance, SSB infestation could dramatically induce the release of (E)-b-caryophyllene [28,37], whereas infestation by WBPH or BPH for 24 h did not ([25]; Fig. 3). In rice, compared to infestation by the chewing herbivore SSB, infestation by the piercing and sucking herbivores WBPH and BPH generally elicited a weaker JA burst but stronger SA and H2O2 bursts [26,37,38]. JA can induce (E)-b-caryophyllene production in several plant species, including rice [27]. Moreover, here we found that JA treatment up-regulated the expression levels of OsCAS, whereas SA treatment moderately inhibited its expression at 8 h after the start of treatment (Fig. 2f). This difference suggests that the different response of OsCAS to SSB and WBPH infestation might be related to the difference in defenserelated signals elicited by the two herbivores. Further studies should address this question. Bioassays found that both female and male WBPH adults preferred feeding on and female adults preferred ovipositing on WT plants to ovipositing on ir-cas plants (Fig. 4). Moreover, there were lower population densities of WBPH on the two ir-cas lines than on WT plants in the field (Fig. 5). Given the fact that ir-cas lines had the same growth phenotype as WT plants [25], the lower population density of WBPH on ir-cas lines, compared to that on WT plants, might demonstrate the impaired emission of (E)-bcaryophyllene. Interestingly, in the laboratory bioassay, we found that the preference of WBPH on XS11 over ir-cas lines gradually decreased (Fig. 4). This is probably due to stronger induced defense responses or poorer nutrient quality at the late stage in WT plants than in ir-cas lines because of higher density of WBPH on the former. In rice, (E)-b-caryophyllene has been reported to be attractive to BPH, T. caelestialium and their natural enemies [25,30,31]. In addition, (E)-b-caryophyllene in maize has also
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Fig. 5. WBPH abundance on wild-type (WT) and ir-cas transgenic plants in the field. Mean number (þSE, n ¼ 3) of WBPH female adults (a), male adults (b) and nymphs (c) on ir-cas lines and WT plants. Letters indicate significant differences among lines within the same date (P < 0.05; one-way ANOVA and Duncan's multiple-range test).
Fig. 4. WBPH preference on wild-type (WT) and ir-cas transgenic plants. (a) Mean number of female adults (þSE, n ¼ 9) and percentage of WBPH eggs (þSE, n ¼ 9) on WT and ir-cas plants in paired-choice assays; (b) mean number of male adults (þSE, n ¼ 9) on WT and ir-cas plants in paired-choice assays. Asterisks indicate significant differences between WT and ir-cas plants (*, P < 0.05; **, P < 0.01; Student's t-test).
recently been proved to be an attractant for two maize pests, the root-feeder D. v. virgifera and the defoliator Spodoptera frugiperda [39], except for functioning as a crucial signal involved in indirect defense of maize plants [4,20]. D. v. virgifera larvae were also discovered to select host plants with a suitable density of conspecifics by perceiving the level of (E)-b-caryophyllene emitted from plants [40]. These results indicate that (E)-b-caryophyllene may play an important role in regulating interactions between plants and insects, a phenomenon warranting future study. Von Arx et al. found that (E)-b-caryophyllene strongly improved sex pheromone-mediated behavioral responses of males of Lobesia botrana [41]. We also observed that both WBPH female and male adults were attracted by (E)-b-caryophyllene (Fig. 4). This compound may also enhance the mating efficiency of WBPH adults. This interesting question should also be explored.
In summary, (E)-b-caryophyllene functions as a host location signal for WBPH. Given that in rice, several insect pests used (E)-bcaryophyllene as a host guiding cue [25,31], it could play a role in pest management in rice. For example, if rice plants that produce (E)-b-caryophyllene are sown at the edges of the field, major insect pests, such as BPH and WBPH, would likely be strongly attracted. If the rest of the plants are engineered to produce no (E)-b-caryophyllene, they will likely remain pest free. In this way, the amount of insecticide applied could be considerably reduced and the environment could be improved. Acknowledgments We thank Emily Wheeler for editorial assistance. This study was jointly sponsored by the Special Fund for Agro-scientific Research in the Public Interest (201403030), the Fundamental Research Funds for the Central Universities (2014FZA6013) and the China Agriculture Research System (CARS-01-21). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pmpp.2015.07.002.
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[23] Y.G. Lou, B. Ma, J.A. Cheng, Attraction of the parasitoid Anagrus nilaparvatae to rice volatiles induced by the rice brown planthopper Nilaparvata lugens, J. Chem. Ecol. 31 (2005b) 2357e2372. [24] J. Lu, H.P. Ju, G.X. Zhou, C.S. Zhu, M. Erb, X.P. Wang, et al., An EAR-motifcontaining ERF transcription factor affects herbivore-induced signaling, defense and resistance in rice, Plant J. 68 (2011) 583e596. [25] Y.T. Xiao, Q. Wang, M. Erb, T.C.J. Turlings, L.Q. Ge, L.F. Hu, et al., Specific herbivore-induced volatiles defend plants and determine insect community composition in the field, Ecol. Lett. 15 (2012) 1130e1139. [26] G.X. Zhou, J.F. Qi, N. Ren, J.A. Cheng, M. Erb, B.Z. Mao, et al., Silencing OsHI-LOX makes rice more susceptible to chewing herbivores, but enhances resistance to a phloem feeder, Plant J. 60 (2009) 638e648. [27] Y.G. Lou, M.H. Du, T.C.J. Turlings, J.A. Cheng, W.F. Shan, Exogenous application of jasmonic acid induces volatile emissions in rice and enhances parasitism of Nilaparvata lugens eggs by the parasitoid Anagrus nilaparvatae, J. Chem. Ecol. 31 (2005a) 1985e2002. [28] J.F. Qi, G.X. Zhou, L.J. Yang, M. Erb, Y.H. Lu, X.L. Sun, et al., The chloroplastlocalized phospholipases D a4 and a5 regulate herbivore-induced direct and indirect defenses in rice, Plant Physiol. 157 (2011) 1987e1999. [29] J.S. Yuan, T.G. Kollner, G. Wiggins, J. Grant, J. Degenhardt, F. Chen, Molecular and genomic basis of volatile-mediated indirect defense against insects in rice, Plant J. 55 (2008) 491e503. [30] A.X. Cheng, C.Y. Xiang, J.X. Li, C.Q. Yang, W.L. Hu, L.J. Wang, et al., The rice (E)beta-caryophyllene synthase (OsTPS3) accounts for the major inducible volatile sesquiterpenes, Phytochemistry 68 (2007) 1632e1641. [31] T. Fujii, M. Hori, K. Matsuda, Attractants for rice leaf bug, Trigonotylus caelestialium (Kirkaldy), are emitted from flowering rice panicles, J. Chem. Ecol. 36 (2010) 999e1005. [32] G.H. Zhou, D.L. Xu, D.G. Xu, M.X. Zhang, Southern rice black-streaked dwarf virus: a white-backed planthopper-transmitted fijivirus threatening rice production in Asia, Front. Microbiol. 4 (2013). [33] Q. Wang, J.C. Li, L.F. Hu, T.F. Zhang, G.X. Zhang, Y.G. Lou, OsMPK3 positively regulates the JA signaling pathway and plant resistance to a chewing herbivore in rice, Plant Cell Rep. 32 (2013) 1075e1084. [34] Z.J. Xin, Z.N. Yu, M. Erb, T.C.J. Turlings, B.H. Wang, J.F. Qi, et al., The broad-leaf herbicide 2,4-dichlorophenoxyacetic acid turns rice into a living trap for a major insect pest and a parasitic wasp, New. Phytol. 194 (2012) 498e510. [35] G. Arimura, K. Matsui, J. Takabayashi, Chemical and molecular ecology of herbivore-induced plant volatiles: proximate factors and their ultimate functions, Plant Cell Physiol. 50 (2009) 911e923. [36] C. Sallaud, D. Rontein, S. Onillon, F. Jabes, P. Duffe, C. Giacalone, et al., A novel pathway for sesquiterpene biosynthesis from Z,Z-farnesyl pyrophosphate in the wild tomato Solanum habrochaites, Plant Cell 21 (2009) 301e317. [37] J. Lu, J. Li, H. Ju, X. Liu, M. Erb, X. Wang, et al., Contrasting effects of ethylene biosynthesis on induced plant resistance against a chewing and a piercingsucking herbivore in rice, Mol. Plant 7 (2014) 1670e1682. [38] B.H. Wang, G.X. Zhou, Z.J. Xin, R. Ji, Y.G. Lou, (Z)-3-hexenal, one of the green leaf volatiles, increases susceptibility of rice to the white-backed planthopper Sogatella furcifera, Plant Mol. Biol. Rep. (2014), http://dx.doi.org/10.1007/ s11105-014-0756-7. [39] C.A.M. Robert, M. Erb, I. Hiltpold, B.E. Hibbard, M.D. Gaillard, J. Bilat, et al., Genetically engineered maize plants reveal distinct costs and benefits of constitutive volatile emissions in the field, Plant Biotechnol. J. 11 (2013) 628e639. [40] C.A.M. Robert, M. Erb, B.E. Hibbard, B.W. French, C. Zwahlen, T.C.J. Turlings, A specialist root herbivore reduces plant resistance and uses an induced plant volatile to aggregate in a density-dependent manner, Funct. Ecol. 26 (2012b) 1429e1440. [41] M. von Arx, D. Schmidt-Busser, P.M. Guerin, Plant volatiles enhance behavioral responses of grapevine moth males, Lobesia botrana to sex pheromone, J. Chem. Ecol. 38 (2012) 222e225.
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Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
(E)-b-caryophyllene functions as a host location signal for the rice white-backed planthopper Sogatella furcifera Qi Wang a, b, Zhaojun Xin a, c, Jiancai Li a, Lingfei Hu a, Yonggen Lou a, Jing Lu a, * a
State Key Laboratory of Rice Biology, Institute of Insect Science, Zhejiang University, Hangzhou 310058, China State Key Lab of Biocontrol, School of Life Sciences, Institute of Entomology, Sun Yat-sen University, Guangzhou 510275, China c Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 December 2014 Received in revised form 24 June 2015 Accepted 4 July 2015 Available online 7 July 2015
Terpenoids are well known to mediate interactions among plants, herbivores and their natural enemies. In rice, (E)-b-caryophyllene has been reported to attract a major insect pest, Nilaparvata lugens, and its egg parasitoid, Anagrus nilaparvatae. Yet, it remains unclear whether (E)-b-caryophyllene also influences other herbivores in rice. Here we demonstrated that a rice (E)-b-caryophyllene synthase, OsCAS, localizes in cytoplasm and that its transcript was up-regulated following mechanical wounding, the infestation of the rice striped stem borer (SSB) Chilo suppressalis and treatment with jasmonic acid. We also revealed that the rice white-backed planthopper Sogatella furcifera preferred feeding and ovipositing on wild-type (WT) plants over feeding on mutants with low levels of (E)-b-caryophyllene. The results indicate that (E)b-caryophyllene may function as an important signal by which herbivores on rice locate their host. © 2015 Hainan Medical University. Published by Elsevier Ltd. All rights reserved.
Keywords: (E)-b-caryophyllene Rice Sogatella furcifera Plant volatile Planteherbivore interaction
1. Introduction Herbivore-induced plant volatile organic compounds (VOCs) play an important role in regulating interactions among plants, herbivores and their natural enemies [1e5]. Terpenoids, the main element of which VOCs are composed, are synthesized via mevalonate (MVA) or 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways in both mono- and dicot plants [6e8]. Terpenoids are known to serve as attractants or repellents to herbivorous insects and their parasitoids and predators [9e12]. Nicotiana attenuata, for example, when attacked by herbivores, releases a considerable amount of cis-a-bergamotene and then recruits carnivores Geocoris pallens and Geocoris punctipes to counter-attack the pest Manduca sexta [13,14]. Similarly, (E)-b-farnesene emitted from Arabidopsis attracts the parasitoid Diaeretiella rapae but has a repellent effect on the aphid Myzus persicae [15]. In addition, terpenoids can be used directly by plants for priming their own defenses before herbivore attack [16e18]. For example, the exogenous application of b-ocimene on Arabidopsis increased the expression of the genes which
* Corresponding author. E-mail addresses: [email protected] (Q. Wang), [email protected] (Z. Xin), [email protected] (J. Li), [email protected] (L. Hu), yglou@zju. edu.cn (Y. Lou), [email protected] (J. Lu).
are responsible for the octadecanoid pathway [19]. These results indicate the complex ecological roles of terpenoids in modulating tritrophic interactions. (E)-b-caryophyllene, a sesquiterpene, has been reported to play an important role in mediating tritrophic interactions. In 2005, for example, Rasmann found that (E)-b-caryophyllene emitted from maize roots strongly attracts an entomopathogenic nematode of the pest Diabrotica virgifera virgifera. Likewise, elevated (E)-b-caryophyllene from leaves infested by Spodoptera litura strongly attracts Cotesia marginiventris, a larval parasitoid of the herbivore [20]. In addition, a recent study also showed that (E)-b-caryophyllene and ethylene were used by D. virgifera virgifera larvae to locate the maize plants which are the most suitable for their development [21]. However, very little is known about how (E)-bcaryophyllene functions in other plant species. Rice, one of the most important food crops in the world, suffers heavily from insect pests, such as piercing-sucking herbivores, brown planthopper (BPH) Nilaparvata lugens and white-backed planthopper (WBPH) Sogatella furcifera, and chewing herbivores, striped stem borer (SSB) Chilo suppressalis and leaf folder Cnaphalocrocis medinalis [22]. Previous studies revealed that herbivore attack induces a set of defensive response in rice by activating the signaling pathways (e.g. jasmonate and ethylene signaling), by expressing defensive genes and by producing both direct (e.g.
http://dx.doi.org/10.1016/j.pmpp.2015.07.002 0885-5765/© 2015 Hainan Medical University. Published by Elsevier Ltd. All rights reserved.
Q. Wang et al. / Physiological and Molecular Plant Pathology 91 (2015) 106e112
trypsin protease inhibitors) and indirect (e.g. VOCs) defense compounds [23e26]. In rice, (E)-b-caryophyllene could be induced by SSB or fall armyworm infestation and treatment with jasmonic acid (JA) but not by BPH infestation [25,27e29]. Using transformed rice plants overexpressing an (E)-b-caryophyllene synthase gene, OsCAS (namely OsTPS3), Cheng et al. showed that (E)-b-caryophyllene attracts Anagrus nilaparvatae [30], an egg parasitoid of rice planthoppers. Moreover, Fujii et al. found that (E)-b-caryophyllene emitted from flowering rice panicles attracts the rice leaf bug Trigonotylus caelestialium [31]. Recently, our study revealed that the constitutively produced (E)-b-caryophyllene in rice is attractive to BPH as well as to its parasitoids and predators [25]. It seems that (E)-b-caryophyllene plays an important role in mediating interactions among rice plants, herbivores and their natural enemies. However, little is known about the effect of (E)-b-caryophyllene on other insects on rice except for the effect on BPH and their natural enemies. In this study, we chose WBPH, a major pest of rice which severely damages rice yield by sucking phloem sap and transmitting the southern rice black-streaked dwarf virus [22,32], as a testing insect and carried out experiments using ir-cas transformed rice plants with silenced expression of OsCAS by the RNAi technology [25]. We asked the following questions: 1) Does (E)-b-caryophyllene influence the host-searching and oviposition behavior of WBPH in the lab? 2) Does (E)-b-caryophyllene affect the population dynamics of WBPH in the field? Our results provide new evidence for the importance of (E)-b-caryophyllene in riceeherbivore interactions.
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third-instar SSB larva that had been starved for 2 h. Nonmanipulated plants were used as controls (C). For the WBPH treatment, plants were individually infested with 15 gravid female adults that were confined in a glass cage (4-cm diameter, 8-cm height, with 48 0.8-mm diameter holes). Control plants were placed in a similar cage without herbivores (Non-infested). For JA and salicylic acid (SA) treatments, plants were individually sprayed with 2 ml of JA (100 mg ml1) or SA (70 mg ml1) in 50 mM sodium phosphate buffer. Control plants were sprayed with 2 ml of the buffer (BUF). 2.4. Subcellular localization of OsCAS A 210-bp sequence was amplified by PCR from the 50 -end of the OsCAS ORF using primers OsCASsp-FP (50 -ATCCATGGCAACCTCTGTTCCGAGTGT-30 ) and OsCASsp-RP (50 -ATCCATGGATTCCTCGGAAACATGAGCT-30 ) (bold letters represent the NcoI digest site). This fragment was inserted into the pEGFP vector (BD Biosciences, San Jose, CA, USA) to fuse it with EGFP. The fusion gene OsCAS::EGFP was then inserted into the transformation vector pCAMBIA1301, yielding pCAMBIA1301-OsCAS::EGFP (Fig. S2A). Meanwhile, the full-length open reading frame (ORF) of EGFP was inserted into pCAMBIA1301, yielding pCAMBIA1301-EGFP (Fig. S2B). These two vectors were used separately to transiently transform leaves of Nicotiana tabacum as described by Zhou et al. [26]. Fluorescence analysis was performed as described by Zhou et al. [26]. 2.5. Gus staining and quantitative GUS activity assay
2. Materials and methods 2.1. Plants and insects Rice genotypes used in this study were wild-type (WT) Xiushui11 (XS11), two ir-cas transgenic lines, L-cas45 and L-cas51 [25] and an OsCASp::GUS reporter line L41 (see below). Generally, hydroponic plants of lines XS11, L41, L-cas45 and L-cas51 with an age of 45e50 days were used for experiments as described by Wang et al. [33]. Some L41 plants were cultivated in soil until earing for organ-specific GUS staining. Colonies of WBPH and SSB were originally obtained from a rice field in Hangzhou, China, and were maintained on Xianyou 63 seedlings. 2.2. Generation and characterization of the OsCASp::GUS reporter line A 2.5-kb genomic DNA region of OsCAS (NCBI: DQ872158), including promoter and transcription initiation regions, was PCRamplified by using primers OsCASp-FP (50 -GCGTCGA0 CATCTGAGTAACAACTTGGGTAA-3 ) and OsCASp-RP (50 -CGAATTCTGAGACTAGCATATCGGTGC-30 ) (bold letters represent digest sites of enzymes SalI and EcoRI, respectively). The amplified fragment was then inserted into the transformation vector pCAMBIA-1391 to fuse it with GUS. Transforming rice, screening homozygous T2 plants and identifying the number of insertions was carried out following Zhou et al. [26]. The homozygous line L41 harboring a single insertion of OsCASp::GUS (Fig. S2) was used for subsequent experiments. 2.3. Plant treatments For the mechanical wounding treatment, rice stems (about 2-cm long of the stems' lower part) were individually damaged using a needle 200 times (W). Control plants (C) were not pierced. For the SSB treatment, plants were individually infested with a single
Plant organs were observed under a microscope after immersion in GUS staining buffer (0.1 M sodium phosphate buffer pH 7.0, 10 mM EDTA, 1 mM X-Gluc and 0.01% Triton X-100) at 37 C for 2e8 h. A quantitative GUS activity assay was performed as described by Xin et al. [34]. Plant tissues were homogenized in extraction buffer (50 mM potassium phosphate, 10 mM EDTA, 0.1% Sarcosyl and 0.1% Triton X-100) and centrifuged. An aliquot of the supernatant was incubated with 4-methylumbelliferyl-b-D-glucuronide (4-MUG) as substrate at 37 C for 30 min. The amount of 4methylumbelliferone (4-MU) product was measured with a DTX880 Multimode Detector (Beckman, Fullerton, CA, USA); another aliquot of the supernatant was used to determine protein concentration according to the Bradford protein assay. 2.6. Quantitative real-time PCR (qPCR) qPCR analysis was performed as described by Wang et al. [33]. Four independent replicates were carried out on XS11 and ir-cas transgenic lines. The following primers and probes were used for qPCR of OsCAS and OsActin (Os03g50885): OsCAS-FP (50 TCGACGCTACGAGATGCTTTTA-30 ), OsCAS-RP (50 -CACCGTAGCAGCTACCTGATCT-30 ), OsActin-FP (50 -TGGACAGGTTATCACCATTGGT-30 ), OsActin-RP (50 -CCGCAGCTTCCATTCCTATG-30 ), OsCAS-Probe (50 CGAGGTGAAATGGCGCTCTGAAGG-30 ) and OsActin-Probe (50 CGTTTCCGCTGCCCTGAGGTCC-30 ). 2.7. Analysis of volatiles Collection, isolation and identification of rice volatiles were carried out as described by Lou et al. [27]. Volatiles emitted from XS11, L-cas45 and L-cas51 plants that were infested with WBPH for 24 h, or non-infested were collected. Quantities for each compound were expressed as percentages of peak areas relative to the internal standard (IS, diethyl sebacate) per 8 h of trapping for one plant. Five independent collections were replicated for each treatment.
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Fig. 1. Localization of OsCAS in Nicotiana tabacum cells. Leaves of N. tabacum were transformed with EGFP and OsCAS::EGFP. After incubation for 36 h, the transformed cells were observed under a confocal microscope. Photographs were taken in UV light (lane 1), visible light (lane 2) and in combination (overlay, lane 3). Fluorescence of EGFP was excited at 488 nm and detected using a 500e530 nm emission filter (lane 1).
2.8. WBPH preference WBPH colonization and oviposition preference was determined as described by Zhou et al. [26]. Fifteen gravid females or 18 male adults were released into a cylinder containing two plants (one WT plant versus one ir-cas line, Fig. S4). The number of WBPH individuals on each plant was recorded at different time points, and the eggs were counted at 72 h post release. The experiment was replicated nine times. 2.9. Field experiment To evaluate the effects of (E)-b-caryophyllene on the population dynamics of WBPH in the field, we carried out an experiment with XS11 and two ir-cas lines in Changxing, Zhejiang, China, in autumn 2010. A rice field consisting of nine plots (3 m 3 m, 225 hills of plants) was used. Each plot was surrounded by a rice buffer zone of 1 m. Three plots were randomly assigned to each line. In total, each line was represented by 675 plants that were assigned to three independent replicate plots. The number of WBPH nymphs, female and male adults in each block was investigated on 28 August and 6 and 14 September. 2.10. Data analysis Differences in the levels of GUS activity, OsCAS transcripts, volatiles and WBPH preference and population were determined using one-way ANOVA or Student's t-test. If the ANOVA was significant, Duncan's multiple range tests were performed to detect significance among groups. In the case of non-normality and/or unequal variances, data were transformed prior to ANOVA. SPSS 17 (IBM, Armonk, NY, USA) was used for all analyses. 3. Results 3.1. Subcellular localization of OsCAS In this study, we investigated the subcellular localization of
OsCAS using a confocal microscopic assay of tobacco cells that were transiently expressed with an OsCAS::EGFP fusion protein. The fluorescence of OsCAS::EGFP was observed in nuclei, cytoplasm and cell membranes (Fig. 1), suggesting that OsCAS is a classic cytosolic sesquiterpene synthase. 3.2. Expression pattern of OsCAS To better understand the transcriptional characterization of OsCAS, we cloned a 2.5-kb promoter sequence of OsCAS, fused it to GUS reporter gene and constructed OsCASp::GUS transgenic rice plants, and finally obtained a homozygous T2 line: L41 (Fig. S1). Using a staining assay, the GUS activity was observed in callus, roots, seedlings, leaf sheaths, stems, leaves and glumes, especially on the cut edges of the vegetative organs, but no activities were detected in mature seeds and stamens (Fig. 2a). A quantitative assay revealed the highest GUS activity in leaves, followed by in spikelets and stems; the lowest activity was found in roots (Fig. 2b). We also found that mechanical wounding induced a short-term elevation of GUS activity, whereas SSB feeding or exogenous JA application induced a long-lasting elevation (Fig. 2, cee). SA treatment, on the other hand, moderately inhibited GUS activity for 4e8 h (Fig. 2f). In addition, qPCR assay showed that transcripts of OsCAS were significantly up-regulated at 48 and 72 h after WBPH infestation (Fig. 2g). Taken together, these results indicate that OsCAS is a tissue-specific gene that is responsive to herbivory. 3.3. Silencing OsCAS decreases the level of (E)-b-caryophyllene but not other volatiles in plants infested by WBPH As expected, WBPH-induced transcript levels of OsCAS in both ir-cas transgenic lines: L-cas45 and L-cas51 were only 17.14 and 21.17% of levels of WT plants (Fig. 3, insert). We next collected and analyzed the volatiles emitted from WT and ir-cas plants that were attacked by WBPH. No difference was observed between WT plants and ir-cas lines in constitutive levels of volatiles except for (E)-bcaryophyllene, which was significantly lower in ir-cas lines than in WT plants (Fig. S3). After one day of WBPH infestation, WT plants
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Fig. 2. Organic and elicited GUS activity in OsCASp::GUS transgenic rice plants. (a) GUS staining of (1) callus, (2) mature seeds, (3) 1-day-old seedlings, (4) 3-day-old seedlings, (5) sheaths at the 4-leaf phase, (6) stems, (7) sheaths, (8) leaves, (9) spikelets (especially in glumes) and (10) stamens; (b) mean GUS activity (þSE, n ¼ 6) of OsCASp::GUS transgenic plants. Letters indicate significant differences among organs (P < 0.05, one-way ANOVA and Duncan's multiple range test). (c) to (f) Mean GUS activity (þSE, n ¼ 4 to 5) of OsCASp::GUS transgenic rice stems that were (a) mechanically wounded, (b) infested by the striped stem borer (SSB), (c) treated with jasmonic acid (JA) or (d) salicylic acid (SA). C, control; BUF, buffer. (g) Mean expression levels (þSE, n ¼ 4) of OsCAS in stems of rice plants infested with the white-backed planthopper (WBPH). Asterisks indicate significant differences in transcript levels between treated and control plants (*, P < 0.05; **, P < 0.01; Student's t-test).
up-regulated the emission of the most abundant monoterpene Slinalool, as well as several major sesquiterpenes, including (E)-abergamotene, sesquisabinene A, b-bisabolene, b-sesquiphellandrene and (E)-g-bisabolene. In contrast, no differences in the amount of (E)-b-caryophyllene from WBPH-infested and from noninfested WT plants were observed (Fig. 3). Again, both ir-cas lines revealed significantly lower emissions of (E)-b-caryophyllene: compared with (E)-b-caryophyllene emissions from WBPHinfested WT plants, emissions of (E)-b-caryophyllene decreased
by 78.32 and 95.45% in L-cas45 and L-cas51 lines, respectively. However, silencing (E)-b-caryophyllene did not influence other monoterpenes and sesquiterpenes (Fig. 3).
3.4. Silencing OsCAS reduces the preference of WBPH We investigated the preference of feeding and ovipositing behavior of WBPH on WT and ir-cas plants. As shown in Fig. 4a, compared with two ir-cas lines, WT plants were more attractive to
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Fig. 3. Analysis of WBPH-induced rice volatiles and expression levels of OsCAS. Relative amounts (þSE, n ¼ 5) of volatiles emitted from wild-type (WT) and ir-cas transgenic plants for 8 h. Insert: Mean expression levels (þSE, n ¼ 4) of OsCAS in WT (XS11) or ir-cas plants infested with WBPH for 24 h. Asterisks indicate significant differences between noninfested and WBPH-infested WT plants (*, P < 0.05; **, P < 0.01; Student's t-test). Letters indicate significant differences among lines under WBPH infestation for 1 day (P < 0.05, one-way ANOVA and Duncan's multiple-range test).
WBPH female adults as places to colonize and on which to oviposit: the numbers of female adults that fed on WT plants were 1.29- and 1.93-fold the number of those that fed on L-cas45 and L-cas51 lines at the start of release; similarly, the numbers of eggs laid on WT plants were 41.85 and 99.07% higher than those laid on L-cas45 and L-cas51 lines, respectively. It is noteworthy that the preference appeared at the start of release and subsequently declined. Additionally, WBPH male adults showed a similar colonization preference for WT plants (Fig. 4b). 3.5. Silencing OsCAS decreases the population density of WBPH in the field We next assessed effects of (E)-b-caryophyllene on WBPH population in the field. In accordance with the preference assay in the lab, our results showed that the population densities of WBPH female and male adults and nymphs on ir-cas lines were lower than the population densities on WT plants, especially in the peak period of the population (Fig. 5). The number of WBPH female adults on L-cas45 and L-cas51 lines was only 48.28 and 38.51% (28 Aug), 50.29 and 31.58% (6 Sep) and 72.88 and 52.54% (14 Sep) of that on WT plants, respectively. Likewise, the population densities of WBPH nymphs in the two ir-cas blocks were reduced by 30e50% compared with those in WT blocks on 28 Aug and 6 Sep. 4. Discussion In plants, monoterpenes are mainly synthesized via the MEP pathway in plastids, whereas sesquiterpenes are mainly synthesized in cytoplasm through the MVA pathway [35,36]. As expected, we found that OsCAS is a cytosolic sesquiterpene synthase (Fig. 1), indicating that (E)-b-caryophyllene in rice is synthesized in cytoplasm. We measured the expression pattern of OsCAS in different organs in rice and found that OsCAS was expressed in many organs, mainly in leaves, followed by in spikelets and stems, with the lowest activity found in roots (Fig. 2). This result was similar to that reported in Cheng et al. [30]. Fujii et al. revealed that during the flowering stage, rice panicles released (E)-b-caryophyllene [31]. We
detected GUS activity in glumes but not stamens, implying that glumes might be the main organ for producing (E)-bcaryophyllene. The transcript level of OsCAS could be induced by wounding and by infestation by SSB or WBPH (Fig. 2c, d and g). However, the response of OsCAS to these treatments was different: fast and shortlived for mechanical wounding, fast and long-lived for SSB infestation, and slow and long-lived for WBPH. This discrepancy corresponded to the result of volatiles released from rice plants. For instance, SSB infestation could dramatically induce the release of (E)-b-caryophyllene [28,37], whereas infestation by WBPH or BPH for 24 h did not ([25]; Fig. 3). In rice, compared to infestation by the chewing herbivore SSB, infestation by the piercing and sucking herbivores WBPH and BPH generally elicited a weaker JA burst but stronger SA and H2O2 bursts [26,37,38]. JA can induce (E)-b-caryophyllene production in several plant species, including rice [27]. Moreover, here we found that JA treatment up-regulated the expression levels of OsCAS, whereas SA treatment moderately inhibited its expression at 8 h after the start of treatment (Fig. 2f). This difference suggests that the different response of OsCAS to SSB and WBPH infestation might be related to the difference in defenserelated signals elicited by the two herbivores. Further studies should address this question. Bioassays found that both female and male WBPH adults preferred feeding on and female adults preferred ovipositing on WT plants to ovipositing on ir-cas plants (Fig. 4). Moreover, there were lower population densities of WBPH on the two ir-cas lines than on WT plants in the field (Fig. 5). Given the fact that ir-cas lines had the same growth phenotype as WT plants [25], the lower population density of WBPH on ir-cas lines, compared to that on WT plants, might demonstrate the impaired emission of (E)-bcaryophyllene. Interestingly, in the laboratory bioassay, we found that the preference of WBPH on XS11 over ir-cas lines gradually decreased (Fig. 4). This is probably due to stronger induced defense responses or poorer nutrient quality at the late stage in WT plants than in ir-cas lines because of higher density of WBPH on the former. In rice, (E)-b-caryophyllene has been reported to be attractive to BPH, T. caelestialium and their natural enemies [25,30,31]. In addition, (E)-b-caryophyllene in maize has also
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Fig. 5. WBPH abundance on wild-type (WT) and ir-cas transgenic plants in the field. Mean number (þSE, n ¼ 3) of WBPH female adults (a), male adults (b) and nymphs (c) on ir-cas lines and WT plants. Letters indicate significant differences among lines within the same date (P < 0.05; one-way ANOVA and Duncan's multiple-range test).
Fig. 4. WBPH preference on wild-type (WT) and ir-cas transgenic plants. (a) Mean number of female adults (þSE, n ¼ 9) and percentage of WBPH eggs (þSE, n ¼ 9) on WT and ir-cas plants in paired-choice assays; (b) mean number of male adults (þSE, n ¼ 9) on WT and ir-cas plants in paired-choice assays. Asterisks indicate significant differences between WT and ir-cas plants (*, P < 0.05; **, P < 0.01; Student's t-test).
recently been proved to be an attractant for two maize pests, the root-feeder D. v. virgifera and the defoliator Spodoptera frugiperda [39], except for functioning as a crucial signal involved in indirect defense of maize plants [4,20]. D. v. virgifera larvae were also discovered to select host plants with a suitable density of conspecifics by perceiving the level of (E)-b-caryophyllene emitted from plants [40]. These results indicate that (E)-b-caryophyllene may play an important role in regulating interactions between plants and insects, a phenomenon warranting future study. Von Arx et al. found that (E)-b-caryophyllene strongly improved sex pheromone-mediated behavioral responses of males of Lobesia botrana [41]. We also observed that both WBPH female and male adults were attracted by (E)-b-caryophyllene (Fig. 4). This compound may also enhance the mating efficiency of WBPH adults. This interesting question should also be explored.
In summary, (E)-b-caryophyllene functions as a host location signal for WBPH. Given that in rice, several insect pests used (E)-bcaryophyllene as a host guiding cue [25,31], it could play a role in pest management in rice. For example, if rice plants that produce (E)-b-caryophyllene are sown at the edges of the field, major insect pests, such as BPH and WBPH, would likely be strongly attracted. If the rest of the plants are engineered to produce no (E)-b-caryophyllene, they will likely remain pest free. In this way, the amount of insecticide applied could be considerably reduced and the environment could be improved. Acknowledgments We thank Emily Wheeler for editorial assistance. This study was jointly sponsored by the Special Fund for Agro-scientific Research in the Public Interest (201403030), the Fundamental Research Funds for the Central Universities (2014FZA6013) and the China Agriculture Research System (CARS-01-21). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.pmpp.2015.07.002.
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