| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online October 26, 2007; 10.1104/pp.107.108811 Plant Physiology 146:974-986 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Silencing Geranylgeranyl Diphosphate Synthase in Nicotiana attenuata Dramatically Impairs Resistance to Tobacco Hornworm1,[W],[OA]Department of Molecular Ecology (A.R.J., K.G., C.H., I.T.B.), and Department of Biochemistry (A.S.), Max Planck Institute for Chemical Ecology, D–07745 Jena, Germany
In bioassays with artificial diets, the 17-hydroxygeranyllinalool diterpenoid glycosides (HGL-DTGs) of Nicotiana attenuata function as antifeedants for the plant's adapted herbivore, tobacco hornworm (Manduca sexta). To determine whether HGL-DTGs have a defensive function in planta, we suppressed HGL-DTG production by silencing the source of the geranylgeranyl diphosphates (GGPPs) required for geranyllinalool biosynthesis, a key intermediate. We used virus-induced gene silencing to suppress transcript levels of GGPP synthase gene (Naggpps) and farnesyl diphosphate (FPP) synthase gene (Nafpps), northern blotting and real-time polymerase chain reaction to quantify transcript accumulations, and radio gas chromatography to analyze prenyltransferase specificity. Silencing Nafpps had no effect on the accumulation of HGL-DTGs but decreased leaf steroid content, demonstrating that DTG-synthesizing enzymes do not use GGPP derived from FPP and confirming FPP's role as a steroid precursor. Unlike plants silenced in the phytoene desaturase gene (Napds), which rapidly bleached, Naggpps-silenced plants had reduced HGL-DTG but not carotenoids or chlorophyll contents, demonstrating that Naggpps supplies substrates for GGPP biosynthesis for HGL-DTGs, but not for phytoene or phytol. Expression of Naggpps in Escherichia coli revealed that the recombinant protein catalyzes the GGPP synthesis from isopentenyl diphosphate and dimethylallyl diphosphate. When fed on silenced plants, hornworm larvae gained up to 3 times more mass than those that fed on empty vector control plants or plants silenced in Nafpps, the trypsin protease inhibitor gene, or the putrescine N-methyltransferase gene. We conclude that HGL-DTGs or other minor undetected diterpenoids derived from GGPP function as direct defenses for N. attenuata and are more potent than nicotine or trypsin protease inhibitors against attack by hornworm larvae.
Plants employ both direct and indirect defenses against herbivores. Direct defenses may provide physical barriers, act to poison herbivores or impede their digestion, or make plant tissues otherwise unappetizing. There are many ways of identifying the metabolites that function as defenses in plants. The effects on insect herbivores of secondary and primary metabolites such as polyphenols, quinones, alkaloids, some saponines, diterpenoid glycosides (DTGs), phytic acid, and trypsin protease inhibitors (TPIs) have been examined by supplementing artificial diets (De Boer and Hanson, 1987  ; Broadway and Duffey, 1988; Felton et al., 1989; Snook et al., 1997; Green et al., 2001; Pohlon and Baldwin, 2001; Jassbi et al., 2006). Unfortunately, some chemicals may be altered in the artificial diets (Usher et al., 1989) and the effectiveness of metabolites as defenses may depend on their chemical context (Felton et al., 1989).
Proteins are thought to be the most important nutrients for Lepidoptera herbivores, and some secondary metabolites, such as tannins, gossypol, and TPIs are thought to inhibit digestion of proteins (Damaty and Hudson, 1975
These complex interactions represent a principal reason why studies of metabolite defensive function in planta are thought to provide more reliable results compared to the use of artificial diet bioassays (Felton et al., 1989
Multiple direct defenses are known to function in the native tobacco N. attenuata; the alkaloid nicotine, a neurotoxin, functions as a potent direct defense, while antidigestive TPIs decrease the nutritional value of plants' tissues and slow insects' growth rates (Steppuhn et al., 2004
DTGs with geranyllinalool (GL) carbon skeletons (GL-DTGs) have been reported to be abundant secondary metabolites in several species of Nicotiana (Shinozaki et al., 1996
Prenyltransferases are key enzymes in the terpenoid biosynthesis pathway. They catalyze the alkylation of one or more (n = 1, 2, 3) molecules of isopentenyl diphosphate (IPP; C5), with an allylic diphosphate such as dimethylallyl diphosphate (DMAPP; C5) to produce geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15), and geranylgeranyl diphosphate (GGPP; C20), respectively (Ohnuma et al., 1998
To investigate the defensive role of DTGs in planta and to determine the biosynthetic pathway of HGL-DTGs in N. attenuata, we used virus-induced gene silencing (VIGS) to suppress the expression of the geranylgeranyl diphosphate synthase (GGPPS) gene (Naggpps) and the farnesyl diphosphate synthase (FPPS) gene (Nafpps). VIGS has been extensively used to investigate the function of genes (Liu et al., 2002 ; Lu et al., 2003; Benedito et al., 2004). It was successfully used to silence both the TPI gene (Natpi) in the leaves and the putrescine N-methyltransferase gene (Napmt) in the roots, suppressing the production of direct defenses TPI and nicotine, respectively (Saedler and Baldwin, 2004). We used Natpi- and Napmt-silenced plants as positive controls for our in planta feeding assays. Phytoene desaturase gene (Napds)-silenced plants bleach rapidly due to suppressed carotenoid production, and these were used as positive controls for the VIGS process (Liu et al., 2002). In addition to its role in the synthesis of diterpenoids, GGPP is also known to be the precursor of carotenoids and the phytol side chain of chlorophylls (Fray et al., 1995; Keller et al., 1998), and we consider the effects of silencing Naggpps on the total chlorophyll and carotenoid contents. Because FPP from Taxus baccata cell cultures is reported to be the substrate for GGPPS (Laskaris et al., 2000), we silenced Nafpps to clarify its role in the biosynthesis of HGL-DTGs.
VIGS of Naggpps and Nafpps Approximately 20 d after inoculating early rosette-stage N. attenuata plants with Agrobacterium tumefaciens GV3101 harboring pTVFPPS, pTVGGPPS, pTV00 (empty vector control [EV]), and pTVPD plasmids, Napds-silenced plants became completely bleached, demonstrating successful gene silencing. To determine the silencing efficiency of Naggpps and Nafpps, total RNA was isolated from the leaves of the transformed plants and subjected to northern analysis (Fig. 2 , insets); transcripts of both genes were substantially decreased in gene specific-silenced lines in comparison to EV controls.
To quantify the silencing efficiency of Naggpps and Nafpps genes, the total RNA of the plants' leaf tissues was reverse transcribed and subjected to SYBR Green real-time (RT)-PCR, using the N. attenuata actin gene (EU273278) as an endogenous control. Using GGPPS-specific primers (Supplemental Table S1), we determined that the relative transcript levels in pTVGGPPS plants (n = 5; 0.015 ± 0.001) were an order of magnitude lower than those in pTVFPPS (n = 5; 0.16 ± 0.009) and EV plants (n = 5; 0.18 ± 0.008; Fig. 2A). When FPPS-specific primers were used, substantially higher levels were observed in EV (n = 5; 0.13 ± 0.01) and pTVGGPPS plants (n = 5; 0.37 ± 0.098) compared to pTVFPPS plants (n = 5; 0.04 ± 0.004; Fig. 2B). The VIGS efficiency (100 – [relative transcript accumulation of silenced line/relative transcript accumulation of EV] x 100) attained in Naggpps- and Nafpps-silenced lines was 92% and 69%, respectively.
Because the silencing efficiency of the Nafpps gene was only 69% and several homologs of fpps and ggpps have been reported in Artemisia tridentata and Arabidopsis (Arabidopsis thaliana; Hemmerlin et al., 2003 After construction of pTVFPPS and pUCFPPS, the Nafpps inserts of at least four clones of each plasmid were sequenced. There was complete identity in the amplified Nafpps sequence between the PCR primers among the clones of each plasmid. For primary cloning of three different Naggpps PCR fragments in pTVGGPPS, pTVGGPPS2, and pUCGGPPS and of the full-length Naggpps on pJETGGPPS, four independent PCRs with eight different primers on cDNA of N. attenuata as template were performed. After successful cloning, the Naggpps inserts of 14 clones (two of pTVGGPPS, two of pTVGGPPS2, six of pUCGGPPS, and four of pJETGGPPS) were sequenced. There were no differences in the amplified sequences between the respective PCR primers among all 14 clones. Because the primers were designed from sequence information from other Solanaceous plants (tomato [Solanum lycopersicum], Capsicum annuum, and N. tabacum), complete identity between primers and template could not be expected. For instance, a comparison of the full-length Solanaceous ggpps sequences available in GenBank reveals an 85% identity of the 3' 900 bp of Naggpps (EF382626) to tomato ggpps (DQ267903) and a 79% identity to C. annuum ggpps (X80267). As a consequence, a few sequence differences between Naggpps and the clones in the primer regions occurred, showing that in our PCR system, primer/Naggpps template mismatches still allowed for efficient PCR amplification. Therefore, the amplification of only one Nafpps and only one Naggpps gene under conditions that allow primer/template mismatches represents strong evidence that there is only one copy of Nafpps and of Naggpps and no family of genes homologous to Nafpps and Naggpps in the genome of N. attenuata. Southern-blot analysis confirmed that the N. attenuata genome harbored only one close homolog of fpps and ggpps (Supplemental Fig. S1, A and B); using a full-length Naggpps probe and a 350-bp Nafpps probe, all four digestions revealed only a single band. Together with the PCR fragment-sequencing results, these results lead us to conclude that there is no other close homolog of Nafpps and Naggpps in N. attenuata.
Radio gas chromatography (GC) analyses revealed that plant protein extracts of pTVGGPPS and pTVFPPS plants contained different levels of prenyltransferase activity. Whereas the extract of the pTVGGPPS line showed mainly FPPS activity (n = 5; 92% ± 2.7% farnesol, and 8% ± 1.9% GG of the total synthesized and hydrolyzed prenyldiphosphate products) and was comparable with the EV plants (n = 5; 98.5% ± 1.7% farnesol, and 1.5% ± 1.2% GG), the protein extract of pTVFPPS plants showed no measurable prenyltransferase activity (Fig. 3 ).
Plant prenyltransferases are only highly similar in their amino acid sequences of the catalytic domain. In a phylogenetic tree of all prenyltransferases comparing the amino acid sequences of the complete open reading frame, FPPS cluster separately from the more related GGPPS or geranyl pyrophosphate synthase (GPPS; Burke and Croteau, 2002 ; Tholl et al., 2004; Bouvier et al., 2005; Schmidt and Gershenzon, 2007b). In contrast to FPPS, where it is possible to predict the putative enzyme activity, functional analyses of GGPPS are necessary to confirm enzyme activity. Because silencing Naggpps did not significantly change the levels of synthesized GGPP in the plants' total protein, we heterologously expressed full-length cDNA in Escherichia coli to determine if the cDNA of Naggpps codes for a functional GGPPS. After the putative chloroplast transit peptide was truncated, NaGGPPS functioned mainly as a GGPPS and showed only a minor amount of FPPS activity. The total synthesized and hydrolyzed prenyldiphosphate products were composed of 85% ± 3.1% GG and 15% ± 2.7% farnesol. The recombinant protein showed no GPPS activity (Fig. 3).
After 14 d of feeding on the VIGS plants, hornworm larvae gained 3- to 4-fold more mass on pTVGGPPS-silenced (n = 30; average mass [AM] = 211.3 ± 24.1 mg; t = 5.259; df = 54; P < 0.0001; 320.3% of the EV plants) than on pTVFPPS-silenced (n = 28; AM = 50.2 ± 6.4 mg; t = 1.337; df = 52; P = 0.187) or EV plants (n = 26; AM = 66.0 ± 10.1 mg; Fig. 4 , white bars). In 40 biological replicates for each silenced line, the larvae fed for 18 d; the number of larvae that survived was as follows: pTVGGPPS, 30 (75%); pTVFPPS, 25 (62.5%); and pTV00, 19 (47.5%).
To determine the effects of silencing Naggpps and Nafpps on secondary metabolite composition, a methanol extract of leaf material from silenced plants was analyzed by reverse-phase (RP)18 HPLC (Supplemental Fig. S2). Levels of HGL-DTGs were lower in pTVGGPPS plants than in pTVFPPS and pTV00 plants (Supplemental Fig. S2; Fig. 4, dotted bars). The average levels of HGL-DTGs in the herbivore-attacked leaves of pTVGGPPS and pTVFPPS plants were 33.6% (n = 10; mean relative area [MRA] % = 99.2 ± 12.3; t = 10.118; df = 18; P < 0.0001) and 101.9% (n = 10; MRA % = 300.7 ± 14.3; t = 0.271; df = 18; P = 0.789) of those measured in EV plants (n = 10; MRA % = 295.1 ± 14.9), respectively (Fig. 4, dotted bars).
To compare the performance of larvae that fed on plants deficient in HGL-DTGs with those deficient in TPIs and nicotine, we repeated the experiments with the addition of Natpi- and Napmt-silenced plants. By day 16, the masses of larvae that fed on pTVGGPPS were significantly (n = 16; AM = 232.9 ± 9.4 mg; t = 7.003; df = 28; P < 0.0001) higher than the masses of those that fed on EV plants (n = 14; AM = 100.2 ± 17.2 mg). Masses of larvae that fed on pTVFPPS (n = 9; AM = 93.3 ± 13.4 mg), pTVPMT (n = 14; AM = 128.2 ± 32.0 mg), and pTVTPI (n = 14; AM = 102.0 ± 17.6 mg) were not significantly different from those that fed on EV plants (Fig. 5A ). As in the first experiment, the number of larvae that survived to the end of the feeding bioassay was higher on pTVGGPPS plants (16, or 59%) than on the EV plants, where only 14 (52%) of 27 larvae survived.
In the second VIGS experiment with additional Napmt- and Natpi-silenced plants, systemic leaf material was analyzed for HGL-DTGs, nicotine, and TPIs (Fig. 5, B–D). Leaves that had not been attacked by larvae were collected at different time points from locations above where the larvae were feeding. The levels of HGL-DTGs in the pTVGGPPS plants were the lowest compared to the other silenced and pTV00 plants at all time points (Fig. 5B). After 11 d of herbivore attack, HGL-DTGs reached their highest levels in all plants (up to 5.5-fold those of EV plants), but levels were still the lowest in pTVGGPPS plants, increasing 3-fold after herbivory. The levels of HGL-DTGs in pTVGGPPS, pTVFPPS, pTVPMT, and pTVTPI plants relative to those in pTV00 plants (n = 6; MRA % = 439.4 ± 11.4) were 32.1% (n = 6; MRA % = 140.9 ± 2.7; t = 25.465; df = 10; P < 0.0001), 64.9% (n = 6; MRA % = 285.3 ± 33.2; t = 4.394; df = 10; P = 0.0013), 63.3% (n = 6; MRA % = 278.1 ± 29.7; t = 5.15; df = 10; P = 0.0004), and 105.4% (n = 6; MRA % = 463.2 ± 29.9; t = 0.743; df = 10; P = 0.4746), respectively (Fig. 5B). By day 17, at the end of the feeding experiment, only levels of HGL-DTGs in pTVGGPPS plants (n = 6; MRA % = 122.4 ± 8.6; t = 7.277; df = 10; P < 0.0001) were significantly different (42.3%; P < 0.0001) from those of the EV plants. Levels of nicotine increased 1.3- to 2.3-fold in the silenced lines and EV plants after 11 d of larval feeding, and silencing Natpi, Naggpps, and Nafpps changed the levels of nicotine to 134.5% (P = 0.001), 80.3% (P = 0.01), and 65.6% (P = 0.0002) of those of the EV. As expected, the nicotine content of the pTVPMT lines was only 16.7% (P < 0.0001) of that in EV controls (Fig. 5C). By day 17, HPLC analyses showed that the lowest levels of nicotine were in the leaves of pTVPMT plants (232.1 ± 21.6 µg g–1 leaf fresh weight [FW]; n = 6; t = 7.311; df = 10; P < 0.0001; 19.1% of EV plants) at the end of the feeding test, while nicotine levels in other silenced plants, pTVGGPPS (1,668.4 ± 205.9 µg g–1 FW; n = 6; 137.9%), pTVFPPS (985.0 µg g–1 FW of leaves; n = 6; 81.4%), and pTVTPI (1,564.7 ± 126.8 µg g–1 FW; n = 6; 129.4%), did not significantly differ from those of pTV00 plants (1,209.6 ± 131.9 µg g–1 FW; n = 6). Analysis of TPI activity revealed similar levels and kinetics of inhibition among pTVGGPPS, pTVFPPS, pTVPMT, and pTV00 plants; as expected, TPI activity was negligible in the pTVTPI plants (Fig. 5D). Like levels of the other defensive substances, levels of TPIs increased 2.3- to 8.3-fold in the silenced and EV plants after 11 d of larval feeding.
To investigate the effect of silencing Nafpps on terpenoid biosynthesis, the head spaces of whole plants were analyzed for volatile sesquiterpenoids. No significant differences (all P values > 0.1) were detected in their levels in comparison to other lines (data not shown). We assumed that Nafpps was involved in steroid biosynthesis, so the concentrations of the free steroids, β-sitosterol and stigmasterol, were measured in dichloromethane extracts of fresh leaves by GC-mass spectrometry (MS). Significant decreases were found in the levels of β-sitosterol (73% of the pTV00 plants; t = 3.099; df = 14; P = 0.0079) and stigmasterol (79% of the pTV00 plants; t = 2.721; df = 14; P = 0.0165) in pTVFPPS plants compared to their levels in EV plants (Fig. 6 ). Silencing Naggpps had no significant effect on the levels of free steroids in pTVGGPPS plants compared to those in EV plants.
Effects of Silencing Prenyltransferases on Photosynthetic Pigments To evaluate the effect of silencing prenyltransferases on photosynthetic pigments, leaf chlorophyll contents were measured during the feeding bioassay with a portable chlorophyll meter (Supplemental Fig. S3). Total extractable chlorophylls (Chl.a + Chl.b) and carotenoids were measured in 80% aqueous acetone extracts of leaves with a UV/VIS spectrophotometer (Supplemental Fig. S4, A and B). Total chlorophyll and carotenoid content did not differ between silenced and pTV00 plants in the same treatment group (Supplemental Figs. S3 and S4). However, the total carotenoid levels of herbivore-attacked plants were significantly lower than in unattacked plants (Supplemental Fig. S4B).
Previous work with N. attenuata plants silenced in their ability to produce nicotine and TPIs suggested that the plants produce additional potent defense metabolites (Steppuhn and Baldwin, 2007 ). When inverted-repeat putrescine N-methyltransferase-silenced N. attenuata plants growing in a field plantation in the Great Basin Desert were elicited by methyl jasmonate (MeJA), the damage these plants received from native herbivores was as low as that of wild-type plants, suggesting the presence of other potent MeJA-inducible defenses (Steppuhn et al., 2004). The results from this study suggest that HGL-DTGs or other minor undetected diterpenoids derived from GGPP are these powerful defenses. Although artificial diet bioassays have indicated that HGL-DTGs have a defensive function (Snook et al., 1997; Jassbi et al., 2006), their defensive function had not been previously explored by manipulating in planta production, the gold standard of proof of defensive function. We silenced two prenyltransferase genes that were potentially involved in HGL-DTG biosynthesis, Naggpps and Nafpps, and compared the growth of hornworm larvae on the silenced lines with their growth on EV plants. In a second study, we used Natpi- and Napmt-silenced plants to compare the antifeedant potential of the well-studied defensive compounds, PIs and nicotine, with that of HGL-DTGs. Because silencing ggpps can reduce the levels of all diterpenoids, we examined the presence of other diterpenoids such as cembrane- and labdane-type diterpenoids, which are found in N. tabacum (Wang et al., 2001, 2004; Wang and Wagner, 2003) by means of GC-MS, HPLC, and NMR analyses of the chloroform extract of the plant, but we could not detect any; the different GL-DTGs were the major diterpenoid constituents of the plant that were extractable by methanol and water (Jassbi et al., 2006). N. attenuata plants, which were inoculated with A. tumefaciens GV3101 carrying pTVFPPS, pTVGGPPS, pTVPMT, and pTVTPI plasmids, grew the same as EV plants. RT-PCR analyses of the total RNA extracted from leaves of the gene-silenced plants revealed that Naggpps and Nafpps are 92% and 69% silenced in their transcript levels, respectively (Fig. 2, A and B). However, the unexpected up-regulation of Nafpps after silencing Naggpps (Fig. 2B) can only be explained when our understanding of the pathways for the synthesis of different metabolites is more complete. Apparently, silencing prenyltransferase synthesis leads to the accumulation of certain metabolites and activates different pathways that increase the level of FPPS transcripts. Radio-GC analyses of the radiolabeled synthesized and hydrolyzed prenyldiphosphates in plants' protein extracts confirmed the reduction of FPPS activity in pTVFPPS plants. Although the levels of synthesized GG were quite low and not significantly reduced after silencing Naggpps, heterologous expression of Naggpps in E. coli and analyses of its expressed product confirmed that NaGGPPS functions exclusively as a GGPPS (Fig. 3). The lower levels of synthesized GG in plant protein extracts indicated that the products of NaGGPPS do not accumulate and are instead rapidly used as a substrate for subsequent reactions such as the synthesis of HGL-DTGs. We found that silencing Naggpps dramatically reduced HGL-DTG levels (Figs. 4, dotted bars, and 5B) but not levels of the other defensive metabolites, nicotine, and PIs (Fig. 5, C and D). Similarly, silencing Napmt or Natpi did not strongly affect the concentration of HGL-DTGs (Fig. 5B). Although the levels of induced HGL-DTGs in pTVFPPS and pTVPMT plants were less than those of EV plants after 11 and 14 d of herbivory (Fig. 5B), the differences in the levels of the suppressed metabolites (HGL-DTGs and nicotine) in the respective silenced plants (pTVGGPPS and pTVPMT) compared to those in EV plants were always significantly higher than those in the other silenced lines (Fig. 5, B and C; P < 0.0001). Silencing Natpi resulted in a line (pTVTPI) that was free of TPI activity. Overall, we conclude that transforming N. attenuata with pTVGGPPS, pTVPMT, and pTVTPI plasmids successfully silenced the production of HGL-DTGs, nicotine, and TPIs, respectively, without affecting the other plant metabolites, including carotenoids and chlorophylls. Therefore, these plants could be used to compare the defensive value of each metabolite in a plant-herbivore interaction. The dramatically increased performance of hornworm larvae feeding on pTVGGPPS plants argues that DTGs are a more effective defense against this herbivore than are TPIs and nicotine.
Although we did not detect significant effects of TPI and nicotine on larval growth in our experiment (Fig. 5A), the defensive function of nicotine and TPIs against hornworm herbivory has been well established by gene-silencing experiments with stably transformed lines of N. attenuata and Nicotiana sylvestris (Voelckel et al., 2001
N. attenuata germinates in the postfire environment and copes with large changes in soil N during postfire succession. As the N content of the soil in natural habitat of N. attenuata remains high for up to 3 years after a fire (Lynds and Baldwin, 1998
The lower levels of HGL-DTGs only in Naggpps-silenced plants suggest that GGPP is a precursor of HGL-DTG diterpenoids in N. attenuata (Fig. 1). In addition to its role in the biosynthesis of diterpenoids and carotenoids, GGPP is required to produce phytol, the side chain of chlorophylls. Carotenoids provide accessory pigments for photosynthesis; when they degrade, plants become photobleached (Fray et al., 1995
Because the amplification of full-length Naggpps and three different fragments of it from N. attenuata cDNA always yielded exactly the same Naggpps sequence and because a Southern blot with chromosomal N. attenuata DNA (Supplemental Fig. S1) suggested the presence of only one copy of Naggpps, we conclude that the N. attenuata genome harbors only one copy of Naggpps. This conclusion does not exclude the existence of additional nonhomologous ggpps genes in N. attenuata or the possibility of complex regulation or compartmentalization of the synthesis of GGPP, as is seen in Arabidopsis and tomato (Bartley and Scolnik, 1995
The ggpps gene in tomato is reportedly induced by both JA and methyl salicylate. However, the product of the gene, (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, is induced mainly by JA (Ament et al., 2006 Silencing Nafpps did not significantly affect the levels of HGL-DTGs, the performance of feeding larvae, or the chlorophyll and the carotenoid contents of the plants (Figs. 4 and 5; Supplemental Figs. S3 and S4), suggesting that NaGGPPS does not use FPP as a substrate and most probably catalyzes the condensation of three IPPs together with one DMAPP to produce GGPP (Fig. 1) and that the FPP produced by the Nafpps gene product is not involved in forming GGPP for carotenoid synthesis. To explore the effect of Nafpps and Naggpps silencing on volatile terpenoids, the headspaces of all of the silenced plants were analyzed, but no significant differences were observed between volatile sesquiterpenoids levels in any lines (data not shown).
The lower levels of free β-sitosterol and stigmasterol in dichloromethane extracts of leaves from pTVFPPS plants confirmed that Nafpps is involved in steroid synthesis in N. attenuata (Fig. 6; Brown, 1998
In summary, we found that silencing Naggpps had the greatest effect on larval performance and we conclude that it is N. attenuata's most effective defense gene against hornworm larvae. The gene product showed only GGPPS activity and synthesizes a precursor of HGL-DTGs. The agreement of these results with prior artificial diet feeding assays confirms the utility of a combination of in planta and bioassay approaches for identifying new defense metabolites. As Naggpps is not involved in primary metabolite synthesis and HGL-DTGs do not incorporate nutrients such as N that commonly limit growth, overexpressing Naggpps could increase the resistance of N. attenuata to hornworm herbivory without affecting the growth of the plant. Given the evidence from this and other studies for a strict separation of prenyltransferases involved in primary metabolism from those involved in secondary metabolism, and given the ubiquity of terpene synthesis, such an approach may also prove useful in agricultural plants. However, as has been shown in N. tabacum where the silencing of a P450 hydroxylase gene suppressed the accumulation of hydroxylated cembranoids (Wang et al., 2001
Construction of Plasmids The primer sequences used are listed in Supplemental Table S1. Cloning was done in Escherichia coli strains TOP10 and TOP10F' (Invitrogen, http://www.invitrogen.com). For the VIGS experiments, pTV00 derivatives were used to transform Agrobacterium tumefaciens strain GV3101.
The Naggpps and Nafpps gene fragments and the full-length Naggpps coding sequence were PCR amplified with proofreading DNA polymerases (VentR DNA Polymerase, http://www.neb.com, or Phusion High-Fidelity DNA Polymerase, http://www.finnzymes.fi) on cDNA from Nicotiana attenuata as template. Primers were designed using sequence information available in GenBank (http://www.ncbi.nlm.nih.gov) from other Solanaceous plants and correspond to the following sequences: GGPPS1-33 and GGPPS2-31 to Nicotiana tabacum GGPPS1 mRNA (AB041632), N. tabacum GGPPS2 mRNA (AB041633), and tomato (Solanum lycopersicum) GGPS2 mRNA (DQ267903); GGPPS3-33 and GGPPS4-31 to Capsicum annuum gene for GGPPS (X80267); GGPPS7-32, GGPPS8-33, and GGPPS11-31 to DQ267903; GGPPS10-26 to DQ267903 and X80267; FPPS1-33 and FPPS2-34 to C. annuum mRNA for FPPS (X84695) and tomato FPPS1 mRNA (AF048747); and FPPS3-34 and FPPS4-33 to N. tabacum mRNA for FPPS (U97330) and X84695. The PCR fragment obtained with primer pair GGPPS10-26 and GGPPS11-31 (1.1 kb) comprised full-length Naggpps (EF382626) and was cloned in pJET1 (3.1 kb; Fermentas, http://www.fermentas.com; DQ317600), yielding pJETGGPPS (4.2 kb). VIGS vector pTVGGPPS (5.9 kb) was obtained by cloning the 0.3-kb PCR fragment synthesized with primer pair GGPPS1-33/GGPPS2-31, comprising positions 491 to 807 of EF382626, in the BamHI and SalI sites of the tobacco rattle virus (TRV)-based plasmid pTV00 (Ratcliff et al., 2001
To silence Nafpps, the PCR fragment obtained with primer pair FPPS3-34 and FPPS4-33 was cloned as a BamHI/SalI fragment in pTV00, leading to silencing vector pTVFPPS (5.9 kb). Plasmid pUCFPPS was constructed by cloning the fragment synthesized with primers FPPS1-33 and FPPS2-34 in the NcoI and PstI sites of pUCPDS5. The GenBank accession numbers of the cloned Nafpps fragments are EF382631 and EF382632. The construction of the vectors pTVTPI (5.7 kb) and pTVPMT (6.2 kb) used for silencing the N. attenuata genes Napmt and Natpi was performed as described previously (pTVPMT2 and pTVPI2 in Saedler and Baldwin, 2004
Smoke-treated N. attenuata seeds originating from glasshouse-grown generations of a collection originating from Utah were germinated on phytagel agar (Sigma, http://www.sigmaaldrich.com) as described previously and transferred to soil after about 10 d (Baldwin et al., 1994
Larvae of the tobacco hornworm (Manduca sexta; Lepidoptera, Sphingidae) were hatched overnight at 28°C from eggs received from Carolina Biological Supply (http://www.carolina.com). One newly hatched larva of hornworm was released on a leaf of each silenced and nonsilenced (empty vector) plant when the corresponding leaf in the Napds-silenced plants (positive control) had completely bleached. A total of 40 and 27 replicate plants from each line were used in the bioassay in the first and second VIGS experiments, respectively (Figs. 4 and 5A). To measure larval performance, larvae were weighed after 4, 14, and 18 d in the first experiment (larval masses after 14 d of feeding are presented in Fig. 4) and after 4, 8, 11, 14, and 16 d of feeding in the second experiment (Fig. 5A). The insect-feeding bioassays were performed in the chambers and under the same conditions as the VIGS experiments. In the first VIGS experiment, Naggpps- and Nafpps-silenced plants were used; in the second experiment, Napmt-, Natpi-, Naggpps-, and Nafpps-silenced plants were used.
Samples were ground in liquid N with a mortar and pestle. Total RNA was extracted with TRI ReagentTM (Sigma) according to the manufacturer's instructions. RNA quality was checked on a denaturing 1% agarose gel (5% formaldehyde) and concentrations were measured spectrophotometrically at 260, 280, and 320 nm. RNA samples were stored at –80°C until use. To determine the efficiency of silencing Naggpps and Nafpps genes, we conducted northern-blot analysis and used quantitative PCR analysis. RNA samples (10 µg, pooled from three individually grown plants) were size fractionated by 1.2% (w/v) agarose-formaldehyde gel electrophoresis and blotted onto a nylon membrane (GeneScreenPlus; PerkinElmer, http://www.perkinelmer.com) according to the manufacturer's protocol. Ethidium bromide fluorescence was used as the loading control. After blotting and UV cross-linking, blots were prehybridized for 1 h at 42°C in Ultrahyb hybridization buffer (Ambion, http://www.ambion.com) followed by overnight hybridization with the 32P-labeled probes. The blots were washed once, 15 min at 60°C with 2x SSC, 0.1% (w/v) SDS, followed by twice at 60°C in 0.1x SSC and 0.1% (w/v) SDS, 20 min each. Blots were exposed for 24 h on a phosphoimage film (Fujifilm, http://www.fujifilm.com). The signals were read by fluorescent image analyzer FLA-3000 (Fujifilm) and quantified with Aida Image Analyzer software (Raytest, http://www.raytest.com). The 313-bp PCR fragment obtained with primer pair GGPPS7-32/GGPPS8-33 and pUCGGPPS as template served as a probe for Naggpps (Supplemental Table S1). The level of Nafpps transcript was detected using probe PCR synthesized with primer pair FPPS1-33/FPPS2-34 and template pUCFPPS (Supplemental Table S1). For RT-PCR analysis, five replicated biological samples were used. One microgram of total RNA obtained from each sample was reverse transcribed using oligo(dT) and Superscript II reverse transcriptase (Invitrogen) for a total volume of 20 µL. cDNA samples were further diluted with water to 40 µL, and 1 µL of the diluted cDNA was used for RT-PCR, carried out on a ABI PRISM 7700 Sequence Detection system (Applied Biosystems, http://www.appliedbiosystems.com) using qPCRTM Core kits (Eurogentec, http://www.eurogentec.com). For each analysis, a linear standard curve, threshold cycle number versus log (designated transcript level), was constructed using a series dilution of a specific cDNA standard; the transcript levels in all unknown samples were determined according to the sta ndard curve. The N. attenuata actin gene was used as an internal standard for normalizing cDNA concentration variations. The sequences of the primers used for SYBR Green-based RT-PCR (the N. attenuata actin gene, Actin-F1 and Actin-R1; Nafpps, FPPS FOR and FPPS REV; Naggpps, GGPPS FOR, GGPPS REV) are provided in Supplemental Table S1.
Genomic DNA was extracted from N. attenuata young leaves using the cetyl-trimethyl-ammonium bromide method (Doyle and Doyle, 1987
Two grams frozen leaf tissue was ground in liquid N using a mortar and pestle and homogenized in extraction buffer containing 50 mM MOPSO, pH 6.8, 5 mM ascorbic acid, 5 mM sodium bisulfite, 5 mM dithiothreitol (DTT), 10 mM MgCl2, 1 mM EDTA, 10% (v/v) glycerol, 1% (w/v) polyvinylpyrrolidone (Mr = 10,000), 4% (w/v) polyvinylpolypyrrolidone, 4% (w/v) Amberlite XAD-4, and 0.1% (v/v) Tween 20. Extracts were shaken for 30 min at 4°C and centrifuged for 30 min at 10,000g. The filtered supernatant (Miracloth) was used for enzyme activity measurements that were carried out for the functional characterization of NaGGPPS.
To express Naggpps in E. coli, a fragment of this gene was amplified using the Expand High Fidelity PCR system (Roche Applied Science, http://www.roche-applied-science.com) with pJETGGPPS as template, thereby removing the coding sequence of the potential chloroplast transit peptide. The cleavage site of this peptide was predicted using the chloroP program (http://www.cbs.dtu.dk). With primer pair GGPPS14-25 and GGPPS13-24, a 0.9-kb fragment was synthesized; after an ATG start codon, the fragment contained codon 54 of EF382626. The fragment was cloned with T-overhangs in plasmid pCR-T7 CT TOPO (2.7 kb; Invitrogen) yielding Naggpps expression vector pGGPPS (3.6 kb). Expression strain E. coli BL 21(DE3) pLys S (Invitrogen) was transformed with pGGPPS. Bacterial cultures expressing Naggpps were grown using Overnight Express Autoinduction system 1 (Novagen, http://www.novagen.com) as described by the manufacturer's instructions but at 18°C. Bacterial pellets were resuspended in assay buffer without DTT and sonicated. The His-tagged recombinant protein was purified with nickel-nitrilotriacetic acid agarose columns (Qiagen, http://www.qiagen.com) according to the manufacturer's instructions. The recombinant protein was eluted with 250 mM imidazole in the assay buffer. After adding DTT, fractions were checked by SDS-PAGE and used to determine enzyme activity.
NaGGPPS assays were carried out in a final volume of 500 µL containing 20 mM 3-(N-Morpholino)-2-hydroxypropanesulfonic acid, pH 7.0, 10 mM of MgCl2, 10% (v/v) glycerol, and 2 mM DTT. Assays were carried out in triplicates of at least three biologically independent experiments with 40 µM [1-14C]IPP (2 MBq/µmol; Biotrend, http://www.biotrend.com) and 40 µM DMAPP (Echelon Biosciences, http://www.echelon-inc.com). After the reaction was initiated by adding recombinant protein, the assay mixture was covered with 1 mL pentane and incubated overnight at 30°C. To stop the assay and hydrolyze all diphosphate esters, a 1-mL solution with 2 units of calf intestine alkaline phosphatase (Sigma) and 2 units of potato (Solanum tuberosum) apyrase (Sigma) in 0.2 M Tris-HCl, pH 9.5 was added to each assay and incubated at 30°C overnight. After enzymatic hydrolysis, the resulting prenyl alcohols were extracted into 2 mL of diethyl ether and, after the addition of a standard terpene mixture (geraniol, farnesol, and geranylgeraniol), the organic extracts were evaporated under N2 and used for radio-GC measurements.
Radio-GC analysis was performed on a Hewlett-Packard (http://www.hp.com) HP6890 gas chromatograph equipped with a thermal conductivity detector (TCD) and Raga radioactivity detector (Raytest) and using a DB5-MS capillary column (J&W Scientific, http://www.jandw.com; 30 m x 0.25 mm i.d., 0.25-µm film thickness). The oven temperature was set 3 min at 70°C, followed by a gradient from 70°C to 240°C at 6°C min–1 and kept 3 min at the final temperature. The carrier gas was H2 with a flow rate of 2 mL min–1. Injector and TCD detector temperatures were maintained at 220°C and 250°C. The injection volume of the concentrated organic phase was 1 µL. The synthesized products, measured by radio-GC, were identified by comparing their retention times with those of coinjected authentic nonradioactive terpene standards, detected via the TCD. Protein concentrations in enzyme assays were measured according to Bradford (Bradford, 1976
Six plants from each line were randomly selected and from each plant six systemic leaves that had not been herbivore attacked were harvested from locations above where the larvae fed. After being frozen in liquid N, leaves (150 mg) were extracted using 1 mL pure methanol or 0.5% acetic acid solution of 40% methanol in an ultrasonic bath for 35 min. Thymol (1 mg/mL, 50 + 800 µL of the methanolic extract) was used as an internal standard and RP18 HPLC analyses were performed as described previously for HGL-DTGs and nicotine (Keinänen et al., 2001
A total of 150 mg of fresh leaf material of the plants was harvested, ground in liquid N, and extracted with 0.3 mL extracting buffer; subsequently, samples were subjected to radial diffusion assays as described previously (van Dam et al., 2001
A total of 300 mg of the systemic leaf plant material, collected 14 d after herbivore treatment in the first VIGS experiment, was extracted as above but with 2 mL dichloromethane. The extracts (nine replicates for each silencing construct) were dried under an N2 stream, redissolved in 100 µL of dichloromethane, and subjected to GC-MS analyses. The GC-MS was carried out on a Varian 3800 gas chromatograph coupled to a Varian Saturn 2000 mass spectrometer operating in EI mode at 70 eV (http://www.varianinc.com). The GC equipped to a DB-5 MS (J&W Scientific column, 30-m x 0.25-mm i.d., 0.25-µm film thickness). The oven temperature was programmed from 285°C to 310°C at 5°C min–1 and kept for 10 min at 310°C. The carrier gas was helium with a flow rate of 1 mL min–1. The injector temperature was set at 310°C in splitless mode. The injection volume was 1 µL for all of the samples. Two major steroids, β-sitosterol and stigmasterol, were identified by comparing their retention times and mass spectrums with those of the authentic standards (from Fluka, http://www.sigmaaldrich.com, and Sigma).
The chlorophyll contents of the plants' leaves were monitored by a portable SPAD-502 chlorophyll meter (Konica Minolta, http://www.konicaminolta.com) and expressed as a SPAD value (Netto et al., 2002
The Student's t tests were performed using the algorithm embedded in StatView (http://www.imp.com). Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers EF 382626, EF 382631, and EF 382632.
The following materials are available in the online version of this article.
We thank David Baulcombe for the gift of pTV00, pTVPD, and pBINTRA; Jianqiang Wu for providing the N. attenuata actin gene primers; Emily Wheeler for editorial assistance; and the glasshouse team for growing the plants. Received September 8, 2007; accepted October 21, 2007; published October 26, 2007.
1 This work was supported by the Alexander von Humboldt Foundation (postdoctoral fellowship to A.R.J.) and by the Max Planck Society.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors is: Ian T. Baldwin (baldwin{at}ice.mpg.de).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.108811 * Corresponding author; e-mail baldwin{at}ice.mpg.de.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOP
ABSTRACT
RESULTS
= 210 nm) relative area percentage of HGL-DTGs (means ± SE, n = 9, area of the peaks at 24–28 min/area of thymols' peak x 100) of the methanolic extract of the leaves from plants not attacked by herbivores. White bars: hornworm larval mass (milligrams; means ± SE; n = 40; in the beginning of the assay) after 14 d of feeding on N. attenuata plants inoculated with A. tumefaciens GV3101 carrying pTV00, pTVGGPPS, or pTVFPPS. ***, Significant differences from pTV00 at P < 0.0001.
