Systemin in Solanum Nigrum. the Tomato-Homologous Polypeptide Does Not Mediate Direct Defense Responses1[W]

Systemin in Solanum nigrum. The Tomato-Homologous Polypeptide Does Not Mediate Direct Defense Responses1[W]

Silvia Schmidt and Ian T. Baldwin* Max Planck Institute for Chemical Ecology, Department of Molecular Ecology, Beutenberg Campus, 07745 Jena, Germany Downloaded from https://academic.oup.com/plphys/article/142/4/1751/6106503 by guest on 27 September 2021

We extend Ryan’s seminal work on the 18-amino acid polypeptide systemin in tomato’s (Solanum lycopersicum) systemic wound response to the closely related solanaceous species Solanum nigrum. We compared wild-type plants to plants transformed with an inverted repeat prosystemin construct (IRSys) to silence the expression of the endogenous S. nigrum prosystemin gene. In wild-type plants elicited with wounding 1 oral secretions from Manduca sexta larvae, trypsin-proteinase inhibitors (TPIs) accumulated even though prosystemin transcripts were down-regulated. Neither reducing the endogenous systemin levels by RNAi nor complementing the plants with systemin by exogenously supplying the polypeptide through excised stems signiﬁcantly increased TPI activity, indicating that systemin and TPIs are not correlated in S. nigrum. The performance of two herbivore species from two feeding guilds, M. sexta larvae and Myzus persicae nicotianae, did not differ between wild-type and IRSys plants, demonstrating that varying endogenous systemin levels do not alter the direct defenses of S. nigrum. Field experiments with wild-type and IRSys plants and the ﬂea beetle Epitrix pubescens supported these glasshouse data. That levels of oral secretion-elicited jasmonic acid did not differ between wild-type and IRSys plants suggests that systemin is unlikely to mediate jasmonate signaling in S. nigrum as it does in tomato. We conclude that the tomato-homologous polypeptide does not mediate direct defense responses in S. nigrum.

Plants not only respond locally to leaf damage between systemin and PIs is supported by the work of caused by wounding, herbivory, or pathogen attack, McGurl et al. (1992), who transformed tomato plants but also induce defenses in distal, unwounded leaves. with an antisense prosystemin construct. A transgenic These systemic defense responses have been exten- line lacking prosystemin was almost completely sup- sively studied in tomato (Solanum lycopersicum), where pressed in its systemic induction of PIs I and II. Fur- an 18-amino acid polypeptide called systemin is known thermore, Manduca sexta larvae that fed on another to play an essential role in generating the mobile transgenic plant silenced in its prosystemin expression wound signal. Systemin is processed from its larger consumed more leaf material and became 3 times precursor, prosystemin, which is synthesized and heavier than those that fed on wild-type plants (Orozco- processed in the vascular phloem parenchyma cells Cardenas et al., 1993). After wounding, transgenic (Narvaez-Vasquez and Ryan, 2004). Constitutive pro- plants transformed to overexpress the prosystemin systemin mRNA expression has been found through- gene constitutively produce PI I and II proteins and out the plant except for the roots (McGurl et al., 1992). accumulate more PIs in local and systemic leaves than After leaf wounding, prosystemin mRNA is induced do wild-type plants (McGurl et al., 1994). Grafting systemically; the highest accumulation is seen after 3 to experiments using these overexpressers as root stocks 4 h (McGurl et al., 1992). Like the prosystemin mRNA, revealed the constitutive production of PIs in wild-type proteinase inhibitor (PI) I mRNA, which encodes for a scions (McGurl et al., 1994), indicating the central role protein with antinutritional effects against several lep- of systemin in generating the systemic wound signal in idopteran herbivores (Johnson et al., 1989; Ryan, 1990), tomato. accumulates systemically after wounding; it is most More recent grafting experiments between jasmonic abundant 8 to 10 h after wounding (McGurl et al., 1992). acid (JA) biosynthesis mutants (called spr2 mutants) Young tomato plants supplied with low concentrations and wild-type plants, or between systemin signaling of systemin through their cut stems accumulated PI I mutants (called spr1 mutants) and wild-type plants, and II (Pearce et al., 1991). This positive correlation showed that both JA biosynthesis and the presence of systemin are required in the local, wounded leaf to produce the systemic signal and hence to induce PIs 1 This work was supported by the Max Planck Society. systemically. On the other hand, neither JA nor sys- * Corresponding author; e-mail [email protected]; fax 49– temin is needed in the systemic, undamaged leaves of (0)3641–571102. tomato plants (Howe, 2004; Schilmiller and Howe, The author responsible for distribution of materials integral to the ﬁndings presented in this article in accordance with the policy 2005). These ﬁndings suggest that systemin acts at described in the Instructions for Authors (www.plantphysiol.org) is: or near the site of wounding by amplifying the JA- Ian T. Baldwin ([email protected]). derived mobile wound signal and are consistent with [W] The online version of this article contains Web-only data. the previously proposed model that places systemin at www.plantphysiol.org/cgi/doi/10.1104/pp.106.089755 the top of the octadecanoid-based signaling pathway

Plant Physiology, December 2006, Vol. 142, pp. 1751–1758, www.plantphysiol.org Ó 2006 American Society of Plant Biologists 1751 Schmidt and Baldwin upstream of JA (Farmer and Ryan, 1992). Evidence that wild-type tissues except for the roots. The sites with systemin levels influence the JA levels of a plant was the highest expression were the flower buds and the provided by Stenzel et al. (2003), who reported a larger leaves, respectively (Fig. 1, A and B). Interestingly, low and more rapid rise in JA levels when leaves of prosystemin mRNA levels were detected in black prosystemin overexpressing plants were wounded berries and stems (Fig. 1, A and B). After a wound- compared to wild-type plants and less JA in plants ing 1 OS treatment, the expression of prosystemin de- transformed with an antisense prosystemin construct. creased rapidly in leaves of wild-type plants and was Chen et al. (2006) also observed 3-fold higher consti- lowest 30 min after elicitation, whereas, in both lines tutive JA levels in prosystemin overexpressing plants transformed with an inverted repeat prosystemin con- compared to wild-type plants. struct (IRSys lines), the expression of prosystemin Using the tomato cDNA as a probe, systemin ho- remained very low (Fig. 2, A and B). mologs have been found in three other solanaceous Downloaded from https://academic.oup.com/plphys/article/142/4/1751/6106503 by guest on 27 September 2021 species (Constabel et al., 1998), including Solanum TPI Accumulation in Wild-Type and IRSys Plants nigrum (black nightshade). S. nigrum is a wild relative of potato (Solanum tuberosum) and tomato (Schmidt To test whether the accumulation of TPI depends on et al., 2005) and has been established as a model the (pro)systemin level of a plant, the amount of TPIs system to study plant-herbivore interactions and their was quantified in uninduced and induced wild-type underlying signaling processes. A PI gene called pin2b and IRSys plants. Although constitutive levels in wild- was found in S. nigrum, which is homologous to and type and transgenic plants were below the detection shares 86% sequence similarity with the tomato piII limit of the assay, levels increased dramatically after gene. S. nigrum has been shown to locally and system- induction (Fig. 3). No significant difference was de- ically induce trypsin-proteinase inhibitor (TPI) activity tected between wild-type and IRSys plants, either in after wounding (Constabel et al., 1998), as well as to local or in systemic leaves. respond systemically by eliciting TPI activity after methyl jasmonate (MeJA) treatment or after attack by Influence of Exogenously Applied Systemin the flea beetle Epitrix pubescens (Schmidt et al., 2004). on TPI Levels S. nigrum systemin, which has 83% amino acid iden- Sys tity to the tomato systemin with the respective prosys- As reducing prosystemin mRNA levels in IR plants did not reduce TPI accumulation, we tried to temins being 81% identical, induced 10 times less PI I when supplied to excised tomato plants than did tomato systemin itself or any of the other systemin homologs (Constabel et al., 1998). On the other hand, S. nigrum plants supplied with S. nigrum systemin did not accumulate more PIs than control plants supplied with buffer despite a difference in the PI transcript levels between both treatments (Constabel et al., 1998). These findings led us to hypothesize that, in S. nigrum, systemin does not mediate direct systemic defense responses as it does in tomato. To test this hypothesis, we posed the following questions. (1) Which tissues of S. nigrum express systemin constitutively? (2) Does S. nigrum induce systemin after treatment with wounding 1 M. sexta oral secretions (OS)? (3) Do PIs accumulate differently in wild-type S. nigrum plants than in plants silenced in their prosystemin expression after a wounding 1 OS elicitation? (4) Does application of systemin induce PI accumulation in S. nigrum? (5) Do herbivores perform differently on wild-type plants than on plants silenced in their prosystemin expression? (6) Do constitutive and induced JA levels differ in S. nigrum wild-type plants from plants silenced in their prosystemin expression?

RESULTS

Spatial and Temporal Prosystemin Transcript Patterns Figure 1. Constitutive prosystemin transcript levels in reproductive (A) and vegetative (B) tissues of wild-type plants. A, Mean 6 SD of five to six Prosystemin, of which at least three genes are pres- pooled samples. B, Mean 6 SD of four to five replicates; LB 5 leaf ent in S. nigrum (Supplemental Fig. S2A), was consti- blade, MR 5 midrib, P 5 petiole, y 5 young, o 5 old. Different letters tutively expressed in all reproductive and vegetative indicate significant differences.

1752 Plant Physiol. Vol. 142, 2006 Solanum nigrum Systemin

[Chrysomelidae]) and phloem sap suckers (i.e. Myzus persicae nicotianae [Aphididae]), were chosen. As measures of the leaf quality of the different genotypes, we quantiﬁed the mass gain of M. sexta, the leaf damage caused by E. pubescens, and the population growth of M. persicae nicotianae, respectively. The measures of M. sexta larval mass and the E. pubescens assay were repeated three times. The data shown in Figure 5 are representative for all three experiments. In none of the three herbivore species was a signiﬁcant difference between wild-type and IRSys lines detected (Fig. 5; all Ps . 0.1240). Downloaded from https://academic.oup.com/plphys/article/142/4/1751/6106503 by guest on 27 September 2021

Influence of Systemin on JA Levels To test whether systemin acts at the top of the octadecanoid pathway upstream of JA, the level of the plant hormone was quantified in the leaves of wild- type plants and IRSys lines treated with wounding 1 OS. The time series of wild-type plants were charac- terized by two peaks 30 min and 3 h after elicitation (Fig. 6, insert). The pattern was similar for IRSys lines, with the second peak more prominent than the first. Neither in uninduced nor in induced leaves were significant differences between wild-type and transgenic lines detected (Fig. 6). The wild-type time series as well as the comparison between wild-type plants Figure 2. Prosystemin transcripts levels in local and systemic leaves of and IRSys lines were repeated twice with similar wild-type plants and IRSys line 1 in the glasshouse (A) and in local results (only one graph is shown). leaves of wild-type plants and IRSys lines 1 and 2 in the field (B) after elicitation with wounding 1 OS. Shown are the mean 6 SE (A) or SD (B) of three to five replicates. DISCUSSION The aim of this study was to determine whether enrich plants with systemin by applying the polypep- systemin’s role in tomato also applies to a solanaceous tide through their cut stems. Applying S. nigrum species that is closely related to tomato. The question systemin or tomato systemin to wild-type S. nigrum was addressed by extending Ryan’s work to S. nigrum plants did not increase TPI levels compared to those of and testing whether systemin mediates direct systemic controls (Fig. 4A). However, the application of MeJA defense responses in this species. To compare trans- was clearly capable of inducing TPIs (Fig. 4A). In to- genic S. nigrum plants silenced in their prosystemin mato plants, the application of tomato systemin sig- expression to wild-type plants, we measured TPI and nificantly increased the level of TPIs compared to control levels, whereas S. nigrum systemin did not (Fig. 4B). Using a MeJA dilution series, we demonstrated that S. nigrum wild-type plants are able to respond to an exogenously applied elicitor in a dose-dependent manner (Fig. 4A, insert). The patterns resulting from treatment of the IRSys lines did not differ significantly from those of treated wild-type plants (Supplemental Fig. S3).

Influence of Systemin Levels on Herbivores of Different Feeding Guilds To evaluate the influence of systemin on direct Figure 3. TPI accumulation in local (L) and systemic (S) leaves after defense mechanisms, we compared the performance wounding 1 OS treatment. Shown are the combined data of two of herbivores on wild-type plants and IRSys lines. independent experiments in which either IRSys line 1 or 2 was Herbivores from two different feeding guilds, namely, compared to wild type (WT). Constitutive levels were below the leaf chewers (i.e. caterpillars of the tobacco hornworm detection limit of the assay in all cases. Bars represent mean 6 SD of M. sexta [Sphingidae] and the flea beetle E. pubescens three to five replicates. Different letters indicate significant differences.

Plant Physiol. Vol. 142, 2006 1753 Schmidt and Baldwin

(pro)systemin levels have an inﬂuence on defense responses, PI levels are typically quantiﬁed as a response variable in tomato. The ability of a transgenic tomato line, silenced in its prosystemin expression, to systemically increase PI I and II (McGurl et al., 1992) was almost completely suppressed, indicating a positive correlation between prosystemin expression and PI accumulation. Com- pared to tomato, the situation is different in S. nigrum. Dramatically reducing levels of prosystemin (in both IRSys lines; Fig. 2, A and B) does not reduce TPI Downloaded from https://academic.oup.com/plphys/article/142/4/1751/6106503 by guest on 27 September 2021

Figure 4. TPI accumulation in leaves of S. nigrum (A) and tomato plants (B) after application of water, S. nigrum systemin, tomato systemin, or MeJA through the cut stems. Insert in A: Dose-dependent accumulation of TPIs in S. nigrum leaves after application of different concentrations of MeJA through the cut petioles. Shown are mean 6 SD of three to ﬁve replicates. Different letters indicate signiﬁcant differences.

JA accumulation after wounding 1 OS treatment. In addition, we compared the performance of three different herbivore species on wild-type and IRSys plants and observed TPI levels in plants supplied with systemin. S. nigrum harbors at least three prosystemin genes (Supplemental Fig. S2A) that are effectively silenced in both IRSys lines (Fig. 2, A and B). The tissue-specific expression pattern of prosystemin in S. nigrum (Fig. 1, A and B) seems to reflect that observed in tomato (McGurl et al., 1992) with high mRNA levels in reproductive and aboveground vegetative tissues. The small amounts of prosystemin mRNA detectable in the black berries (as opposed to the green berries) and Figure 5. Herbivore performance on wild-type and IRSys plants. A, roots suggest that systemin is unimportant in these Mass of M. sexta caterpillars reared on wild-type and IRSys plants. tissues. While the levels of constitutive prosystemin Shown are mean 6 SD of 19 to 22 larvae feeding individually on the transcript in S. nigrum match the levels reported from respective genotypes (repeated-measures ANOVA: P 5 0.51). B, Mean tomato, the picture changes after induction. In tomato, leaf damage caused by E. pubescens feeding on field-grown wild-type plants and IRSys lines. Shown are mean 6 SD of 15 plants per genotype prosystemin is systemically induced after wounding (Bonferroni-corrected Wilcoxon signed rank test for wild type singly (McGurl et al., 1992). In contrast, in S. nigrum local and compared to each IRSys line at each day: P . 0.025). C, Number of M. systemic transcript levels rapidly decrease after OS persicae nicotianae aphids after 10 d starting from one female placed elicitation under both glasshouse as well as field on each of 15 plants of each genotype. Shown are mean 6 SD. Different conditions (Fig. 2, A and B). To demonstrate that the letters indicate significant differences.

1754 Plant Physiol. Vol. 142, 2006 Solanum nigrum Systemin

Figure 6. JA content in leaves of wild-type plants and IRSys lines after wounding 1 OS treatment. Shown are mean 6 SD of ﬁve individual plants per time point and genotype (single ANOVA followed by LSD post- hoc test per time point: P . 0.05). Insert: Shown are mean 6 SE of ﬁve individual wild-type plants per time point. Downloaded from https://academic.oup.com/plphys/article/142/4/1751/6106503 by guest on 27 September 2021

(Fig. 3). In addition, TPI levels in wild-type plants in- We applied tomato systemin to tomato plants, with the crease while prosystemin transcript levels decrease expected result: Levels of TPIs significantly increased after elicitation. This demonstrates an absence of cor- compared to control levels (Fig. 4B). However, apply- relation between systemin and TPIs in S. nigrum. The ing S. nigrum systemin to tomato did not (Fig. 4B), observed positive correlation in tomato, however, which was consistent with the findings of Constabel could theoretically be due to an insertion effect of the et al. (1998), who pointed out that, in tomato, S. nigrum transgene, as only the F1 progeny of a single primary systemin is 10 times less effective than tomato system- tomato transformant was tested. Working with trans- in in eliciting PI I. Supplying both S. nigrum IRSys lines formed plants always carries the risk of detecting a with water or S. nigrum systemin did not lead to any transgene-insertion effect rather than a gene-of-interest significant differences compared to the wild-type effect; this risk is greatly reduced when the phenotype plants (Supplemental Fig. S3), supporting the absence occurs in more than one independently transformed of a correlation between systemin and TPIs in S. line. Figure 3 shows the combined data from two inde- nigrum. pendent experiments in which either IRSys line 1 or To test the influence of endogenous systemin levels IRSys line 2 was compared to wild type. Even though on S. nigrum’s resistance to insect attack, the perfor- the absolute TPI levels differed between the two exper- mance of different herbivores on wild-type and IRSys iments, in none of the experiments did TPI levels differ plants was evaluated. Larvae of the tobacco hornworm significantly between transgenic and wild-type plants. M. sexta, a Solanaceae specialist, gained the same mass To increase endogenous systemin levels, plants were when reared on the different genotypes (Fig. 5A). Dra- supplied with additional systemin by applying the matic differences in weight gain have been reported polypeptide through the cut stems or petioles. In S. for tomato plants overexpressing the prosystemin nigrum wild-type plants, neither S. nigrum systemin gene compared to wild-type tomato plants (Orozco- nor tomato systemin was able to elicit higher TPI levels Cardenas et al., 1993). Nevertheless, data derived from compared to those of water-treated controls (Fig. 4A). these overexpressor lines need to be interpreted cau- The former phenomenon was reported previously by tiously as the ectopic expression of prosystemin Constabel et al. (1998), which suggests that the exci- mRNA driven by an 35S promoter produces systemin sion itself activated the wound response to maximal in tissues that normally do not express the gene, for levels, making it difficult to discern whether the PI example, roots (McGurl et al., 1992). The flea beetle E. accumulation was affected by exogenously applied pubescens, another Solanaceae specialist, has been re- systemin. By using MeJA as a positive control (Fig. peatedly observed to feed on S. nigrum at our field site. 4A), we demonstrated that this was not the case but The mean damage inflicted by this species on the IRSys that excised S. nigrum plants supplied with MeJA are lines did not differ significantly from that done to able to significantly increase TPI levels over control wild-type plants (Fig. 5B). Finally, the population levels in a dose-dependant manner (Fig. 4A, insert). growth of M. persicae nicotianae did not differ between Constabel et al. (1998) hypothesized that S. nigrum genotypes (Fig. 5C). Taken together, these results systemin mediated defense gene induction in S. nig- demonstrate that the quality of IRSys and wild-type rum because systemin treatments increased PI-mRNA leaves does not differ for these herbivores. accumulation more than did treatment with buffer To test whether the endogenous systemin levels only, even though this differential transcriptional ex- influence a plant’s ability to produce JA, the plant pression was not reflected on the protein level. Our hormone was quantified in both uninduced and OS- analysis of S. nigrum does not support this hypothesis. elicited wild-type and IRSys plants. Wild-type plants

Plant Physiol. Vol. 142, 2006 1755 Schmidt and Baldwin clearly responded to the treatment (Fig. 6, insert), copy of the transgene. Southern-blot analysis (Supplemental Fig. S2B) resulted showing a two-peaked pattern of JA accumulation. in two independently transformed inverted repeat lines. Seeds of these lines were used for all experiments and will be made freely available to academic The amount of JA did not differ significantly between investigators for noncommercial research purposes. Line S03-71-13 and line wild-type and IRSys plants (Fig. 6), indicating little or S03-82-3 are referred to as IRSys line 1 and IRSys line 2. no correlation between systemin and JA in S. nigrum. Given that prosystemin transcripts are down-regulated Southern-Blot Analysis after induction (Fig. 2), systemin and JA may be neg- atively correlated in S. nigrum, but the low constitutive Genomic DNA of wild-type and IRSys plants was isolated from S. nigrum leaves using a modified cetyl trimethyl ammonium bromide method (Rogers JA levels in both IRSys lines argue against this. These and Bendich, 1994) as described by Bubner et al. (2004). The final DNA pellet data suggest that systemin in S. nigrum does not act was rehydrated in 50 mLof13 Tris/EDTA buffer (10 mM TrisCl, pH 8, 1 mM at the top of the octadecanoid-based signaling path- EDTA, pH 8). Restriction digests were done using BamHI, EcoRI, and EcoRV way upstream of JA as has been proposed for tomato for the wild-type DNA and EcoRV or XbaI for the DNA of the two IRSys lines. Downloaded from https://academic.oup.com/plphys/article/142/4/1751/6106503 by guest on 27 September 2021 (Farmer and Ryan, 1992). Current knowledge about Plasmids (pSOL3SYS1) were linearized using BamHI, XhoI, or XbaI. Size fractionation by 0.8% agarose gel electrophoresis was followed by Southern systemin based on research in tomato appears not to blotting onto a nylon membrane with a high-salt buffer (Brown, 1995). The apply to a closely related member of the same family. blots were hybridized with 32P-labeled probes specific for the prosystemin The possibility that systemin might play completely gene (primer pair SYS5-32 [5#-GCGGCGCCATGGTCTGTCTGCATTTTGGG- AGG-3#] and SYS6-31 [5#-GCGGCGCTGCAGTGGAACATGAGGAGGG- different roles even in closely related species is sup- # ported by Boller’s (2005) analysis of systemin se- AGG-3 ]) or the hygromycin resistance gene hptII. quences, which concluded that systemin appears to be under diversifying selection. Plant Treatments To mimic herbivore feeding, one leaf was wounded with a fabric pattern wheel, causing three rows of punctured wounds on each side of the midrib. MATERIALS AND METHODS The wounds were immediately supplied with OS of Manduca sexta larvae. OS were collected from third- to fourth-instar larvae hatched from eggs (Carolina Plant Growth Biological Supply) and reared on S. nigrum wild-type plants. OS were diluted 1:1 (v/v) with deionized water prior usage. For the prosystemin expression The Solanum nigrum inbred line Sn30 (Schmidt et al., 2004) was used as a time series, the PI assay, and the JA measurements, a fully expanded leaf of the wild-type control for all experiments. To synchronize germination, seeds of main axis (normally one leaf at nodes six to eight) was induced as described wild-type and transgenic plants were incubated in 5 mL of 1 M KNO3 above. After the respective time points (for the PI assay, after 72 h), the locally supplemented with 50 mL of 0.1 M gibberellic acid (Roth) and 25 mL of 0.5% treated leaf and in the case of the prosystemin time series the uninduced leaf (v/v) Tween 20 (Merck) at 4°C overnight. Seeds were germinated in Teku pots one node above the treated leaf were harvested, flash frozen in liquid nitrogen, with a peat-based substrate (Klasmann Tonsubstrat) and transferred to 9- 3 and stored at 280°C until further processing. As the plants grew slower in the 9- 3 9.5-cm pots containing the same substrate after about 14 d. At all stages field than in the glasshouse, in the field experiment the leaf at the fourth node the plants were grown in the greenhouse (16 h light, supplemental lighting by was treated and harvested as described above. To measure the constitutive Philips Master Sun-T PIA Agro 400 and Sun-T PIA Plus 600 W sodium lights/ prosystemin expression in different plant tissues, the whole plant remained 23°Cto25°C/45% to 55% humidity; 8 h dark/19°Cto23°C/45% to 55% untreated and RNA was extracted from old (third node) leaf blades, petioles, humidity) of the Max Planck Institute for Chemical Ecology (Jena, Germany). and midribs; young (seventh node) leaf blades, petioles, and midribs; and The procedure was the same for tomato (Solanum lycopersicum cv Castlemart), stems, roots, buds, flowers, and green and black berries. except that the seeds were soaked in water at 4°C overnight. The PI-inducing effect of S. nigrum and tomato systemin was tested by Plants used in the two field experiments in July and August 2005 (flea excising S. nigrum and tomato wild-type plants at the base of the stem beetle herbivory and prosystemin expression after wounding 1 OS treatment) according to Pearce et al. (1993) with a scalpel and placing them into tubes were planted at the field site in Dornburg (north of Jena, Germany) 24 or 21 d containing 500 mL of water either pure or supplemented with 2.5 pmol postsowing after being acclimatized to outdoor conditions for 3 to 5 d. The systemin (AnaSpec; according to protein sequences published by Constabel release of transformed plants at the Dornburg field site was conducted in et al., 1998) or 150 mg of MeJA as a positive control. After the complete solution compliance with EU and German regulations (release application nos. 6786– had been taken up, plants were transferred to water for 72 h and the leaf at the 01–0156 [IRSys line 1] and 6786–01–0165 [IRSys line 2]) as administered by the sixth (S. nigrum) or the third node (tomato) was harvested, flash frozen in Thu¨ ringer Landesverwaltungsamt and the Thu¨ ringer Landesamt fu¨ r Leben- liquid nitrogen, and stored at 280°C until processing for TPI assay. The smittelsicherheit und Verbraucherschutz. experiment was complemented by supplying IRSys leaves with pure water or All experiments were conducted with 4- to 5-week-old plants except those water supplemented with 2.5 pmol systemin or 150 mg of MeJA through their involving the harvesting of reproductive tissues. cut petioles. To demonstrate that excised S. nigrum leaves are capable of responding to exogenously applied MeJA in a dose-dependent manner, S. nigrum wild-type leaves were supplied with different concentrations of MeJA IRSys Lines in 500 mL of water through their cut petioles followed by the procedure described above. Transgenic S. nigrum lines silenced in their prosystemin expression were All experiments described above were based on four to six individual constructed using the silencing vector pSOL3SYS1 (Supplemental Fig. S1), plants for each treatment and/or harvest time point. The individual repro- which is based on the pSOL3RCA silencing vector described in detail by ductive tissues samples were pooled out of 15 buds, five flowers, three green Bubner et al. (2006). The original RCA inverted repeat fragments were berries, and two black berries per plant, respectively. consecutively replaced by two 399-bp fragments of the S. nigrum prosystemin gene after being PCR amplified using the primer pairs SYS5-32 (5#-GCG- GCGCCATGGTCTGTCTGCATTTTGGGAGG-3#) and SYS6-31 (5#-GCGGC- Herbivore Experiments GCTGCAGTGGAACATGAGGAGGGAGG-3#), and SYS7-32 (5#-GCGGCGC- TCGAGTCTGTCTGCATTTTGGGAGG-3#) and SYS8-32 (5#-GCGGCGGAG- M. sexta larvae were reared from eggs (obtained from the M. sexta colony at CTCGTGGAACATGAGGAGGGAGG-3#), respectively, based on the mRNA the Max Planck Institute for Chemical Ecology in Jena, Germany) and one sequence published by Constabel et al. (1998). Agrobacterium tumefaciens- neonate was placed on each of the 25 individual plants per genotype (wild mediated transformation was conducted as described by Kru¨ gel et al. (2002), type and both IRSys lines). The caterpillars were weighed after 3, 5, 9, and 11 d. and T1 (transformation generation 1) plants homozygous for the transgene Naturally occurring adult flea beetles (Epitrix pubescens) were allowed to were selected by a hygromycin resistance screen of their progeny. The progeny feed on 45 field-grown plants planted as triplicates of the three genotypes over of homozygous T1 plants was additionally tested to see if it harbored a single a period of 10 d. The damage done to the plant was recorded on days 2, 4, and

1756 Plant Physiol. Vol. 142, 2006 Solanum nigrum Systemin

10. To quantify the damage, each individual leaf was categorized according to psi). The most abundant and characteristic fragment ion was chosen for the following damage classes: 0 5 0% damage, 1 5 1% to 5% damage, 2 5 6% quantiﬁcation. to 10% damage, 3 5 11% to 25% damage, 4 5 26% to 50% damage, and 5 5 The amount of JA per sample was calculated with the following formula 51% to 100% damage. Based on the damage level estimated for each individ- [(peak area endogenous JA 3 200 ng mL21) 3 peak area ISTD21] and related ual leaf, a mean value was calculated for the entire plant. As these mean plant to 1 mg leaf tissue. data are noncontinuous percentage values and the damage experienced by wild-type plants was compared singly to the damage done to each IRSys line, thus having two analyses per recording day, Bonferroni-corrected Wilcoxon Statistics signed rank tests were performed. Data were analyzed by ANOVA followed by an LSD post-hoc test. To ensure Myzus persicae nicotianae aphids were collected on Nicotiana attenuata plants homogeneity of variances data were transformed if necessary (square root: in our greenhouse and transferred to S. nigrum, where they were allowed to Figs. 1A and 2A; LG10: Figs. 3, left, 4A, 7C, and, partly, 8B; reciprocal: Figs. 3, establish a population for about 2 weeks. Single females of this population right, and 6). In cases in which variances could not be homogenized by were used to infest 15 S. nigrum plants of each genotype (wild type and both transformation, a Welch test followed by a Dunnett T3 test was performed

IRSys lines), and after 10 d the population size on each plant was counted. Downloaded from https://academic.oup.com/plphys/article/142/4/1751/6106503 by guest on 27 September 2021 (Figs. 1B, 2B, and 5A). The M. sexta caterpillar mass data were analyzed using repeated-measures ANOVA. All analyses were done using the software RNA Extraction and Quantitative Real-Time PCR package SPSS.

Harvested tissues were ground individually in liquid nitrogen and total Supplemental Data RNA was isolated following a modified TRI Reagent procedure for polysac- charide- and proteoglycan-rich sources (The Institute for Genomic Research, The following materials are available in the online version of this article. 2003). Total RNA (100 ng) was reverse transcribed into cDNA using MultiScribe Supplemental Figure S1. Silencing vector pSOL3SYS1. reverse transcriptase (Applied Biosystems) according to the manufacturer’s instructions. Prosystemin mRNA expression was quantified by real-time PCR Supplemental Figure S2. Southern blots of wild type and both IRSys (ABI PRISM 7700 sequence detection system; Applied Biosystems) using the lines. quantitative PCR core reagent kit (Eurogentec), a specific TaqMan primer pair, and a double fluorescent dye-labeled TaqMan probe specific for the S. nigrum Supplemental Figure S3. TPI accumulation in leaves of S. nigrum wild- prosystemin gene based on the sequence published by Constabel et al. (1998). type plants and both IRSys lines after application of water, S. nigrum The probe was designed to detect only the endogenous prosystemin gene and systemin (S.n.sys), or MeJA through the cut petioles. not the region selected for the inverted repeat construct. The relative target gene expression of each sample was determined using standard curves. cDNA for these standards was obtained by reverse transcription of S. nigrum RNA extracted from a wounding 1 OS-treated wild-type plant using SuperScript II ACKNOWLEDGMENTS RNaseH2 reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. With each measurement, TaqMan reactions of four defined cDNA We dedicate this paper to Clarence ‘‘Bud’’ Ryan, whose research program dilutions (20 ng/mLto0.02ng/mL) were run using the primers and probe inspired more than three decades of research into herbivore-induced de- described above. Ct values (the cycle number C at which the PCR product fenses. We thank K. Gase, W. Kro¨ber, A. Wissgott, and S. Kutschbach for the triggers a certain amount of fluorescence [threshold t]) of the four standards transformation construct and for plant transformation, A. Berg and E. Rothe were plotted against the respective cDNA concentration and the obtained curve for assistance with the JA analysis, J. Kellmann for managing the onerous was used to relatively calculate the transcript amount of the samples. legal obstacles to conducting field work with genetically modified plants in Germany, G. Howe for kindly providing us S. lycopersicum seeds, and C. Kost, M. Hartl, C. Voelckel, T. Kaiser, and two anonymous reviewers for critically TPI Assay commenting on earlier versions of the manuscript.

Harvested leaves were ground in liquid nitrogen individually and total Received September 12, 2006; accepted October 22, 2006; published October protein was extracted using 2 mL of protein extraction buffer (Jongsma et al., 27, 2006. 1994) per milligram plant tissue, followed by vortexing and centrifugation as described in Van Dam et al. (2001). Protein content of the samples was determined in technical triplicate using the method of Bradford (1976) with an IgG-based (Sigma) standard curve. TPI was determined by the radial immu- LITERATURE CITED nodiffusion assay (Jongsma et al., 1993) using a soybean trypsin inhibitor (Sigma) dilution series as a standard. The final TPI activity is expressed as Boller T (2005) Peptide signalling in plant development and self/non-self nmol mg21 total protein. perception. Curr Opin Cell Biol 17: 116–122 Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye bind- JA Measurements ing. Anal Biochem 72: 248–254 Brown T (1995) Southern blotting. In F Ausubel, R Brent, RE Kingston, DD Approximately 300 mg of harvested leaf tissue were homogenized in 1 mL Moore, JG Seidman, JA Smith, K Struhl, Short Protocols in Molecular 21 13 of ethyl acetate spiked with 200 ng mL methanolic [ C2]JA as an internal Biology. Wiley, New York, pp 2128–2132 standard. After centrifugation at 13,000 rpm for 20 min at 4°C, the supernatant Bubner B, Gase K, Baldwin IT (2004) Two-fold differences are the detec- was transferred to another tube and the extract was redissolved in 1 mL of tion limit for determining transgene copy numbers in plants by real- ethyl acetate. Following another centrifugation step, the combined superna- time PCR. BMC Biotechnol 4: 14 tants were evaporated and the dried sample was redissolved in 500 mLof70% BubnerB,GaseK,BergerB,LinkD,BaldwinIT(2006) Occurrence of (v/v) methanol. After vortexing for 5 min, the sample was centrifuged for tetraploidy in Nicotiana attenuata plants after Agrobacterium-mediated 10 min at 13,000 rpm and 15 mL of the supernatant was analyzed using a transformation is genotype specific but independent of polysomaty of Varian 1200 L triple quadrupole mass spectrometer. explant tissue. Plant Cell Rep 25: 668–675 For the HPLC, a Pursuit C8 column (150 mm 3 2.0 mm, 3-mm particle size) Chen H, Jones AD, Howe GA (2006) Constitutive activation of the was used and a gradient of water and methanol, both including 0.05% (v/v) jasmonate signaling pathway enhances the production of secondary formic acid, was run as the mobile phase with a flow rate of 0.2 mL/min. The metabolites in tomato. FEBS Lett 580: 2540–2546 mass spectrometer was run in negative electrospray ionization mode with an Constabel CP, Yip L, Ryan CA (1998) Prosystemin from potato, black argon pressure of 0.279972 Pa (52.1 mTorr) in the collision cell. The mass nightshade, and bell pepper: primary structure and biological activity of spectrometer was set up with a capillary voltage of 23,200 V, a shield voltage predicted systemin polypeptides. Plant Mol Biol 36: 55–62 of 600 V, and a detector voltage of 1,800 V. The pressure of the drying gas was FarmerEE, Ryan CA (1992) Octadecanoid precursorsof jasmonic acidactivate the 131,005 Pa (519 psi) at 300°C; that of the nebulizing gas was 379,225 Pa (555 synthesis of wound-inducible proteinase-inhibitors. Plant Cell 4: 129–134

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Howe GA (2004) Jasmonates as signals in the wound response. J Plant substituted systemin, an 18-amino acid polypeptide inducer of plant Growth Regul 23: 223–237 defensive genes. J Biol Chem 268: 212–216 Johnson R, Narvaez J, An GH, Ryan C (1989) Expression of proteinase Pearce G, Strydom D, Johnson S, Ryan CA (1991) A polypeptide from inhibitor-I and inhibitor-II in transgenic tobacco plants: effects on tomato leaves induces wound-inducible proteinase inhibitor proteins. natural defense against Manduca sexta larvae. Proc Natl Acad Sci USA Science 253: 895–898 86: 9871–9875 Rogers S, Bendich A (1994) Extraction of Total Cellular DNA from Plants, Jongsma MA, Bakker PL, Stiekema WJ (1993) Quantitative determination Algae and Fungi, Ed 2. Kluwer Academic Publishers, Dordrecht, The of serine-proteinase inhibitor activity using a radial diffusion assay. Netherlands Anal Biochem 212: 79–84 Ryan CA (1990) Protease inhibitors in plants: genes for improving defenses Jongsma MA, Bakker PL, Visser B, Stiekema WJ (1994) Trypsin-inhibitor activity against insects and pathogens. Annu Rev Phytopathol 28: 425–449 in mature tobacco and tomato plants is mainly induced locally in response to Schilmiller AL, Howe GA (2005) Systemic signaling in the wound re- insect attack, wounding and virus-infection. Planta 195: 29–35 sponse. Curr Opin Plant Biol 8: 369–377 Kru¨ gel T, Lim M, Gase K, Halitschke R, Baldwin IT (2002) Agrobacterium- Schmidt DD, Kessler A, Kessler D, Schmidt S, Lim M, Gase K, Baldwin

mediated transformation of Nicotiana attenuata,amodelecological IT (2004) Solanum nigrum: a model ecological expression system and its Downloaded from https://academic.oup.com/plphys/article/142/4/1751/6106503 by guest on 27 September 2021 expression system. Chemoecology 12: 177–183 tools. Mol Ecol 13: 981–995 McGurl B, Orozco-Cardenas M, Pearce G, Ryan CA (1994) Overexpression Schmidt DD, Voelckel C, Hartl M, Schmidt S, Baldwin IT (2005) Spec- of the prosystemin gene in transgenic tomato plants generates a sys- ificity in ecological interactions. Attack from the same lepidopteran temic signal that constitutively induces proteinase inhibitor synthesis. herbivore results in species-specific transcriptional responses in two Proc Natl Acad Sci USA 91: 9799–9802 solanaceous host plants. Plant Physiol 138: 1763–1773 McGurl B, Pearce G, Orozco-Cardenas M, Ryan CA (1992) Structure, Stenzel I, Hause B, Maucher H, Pitzschke A, Miersch O, Ziegler J, Ryan expression, and antisense inhibition of the systemin precursor gene. CA, Wasternack C (2003) Allene oxide cyclase dependence of the Science 255: 1570–1573 wound response and vascular bundle-specific generation of jasmonates Narvaez-Vasquez J, Ryan CA (2004) The cellular localization of prosyste- in tomato: amplification in wound signalling. Plant J 33: 577–589 min: a functional role for phloem parenchyma in systemic wound The Institute for Genomic Research (2003) RNA Isolation Using Trizol, signaling. Planta 218: 360–369 Online Protocol. http://www.tigr.org/tdb/potato/images/SGED_ Orozco-Cardenas M, McGurl B, Ryan CA (1993) Expression of an anti- SOP_3.1.1.pdf (April 18, 2003) sense prosystemin gene in tomato plants reduces resistance toward Van Dam NM, Horn M, Mares M, Baldwin IT (2001) Ontogeny constrains Manduca sexta larvae. Proc Natl Acad Sci USA 90: 8273–8276 systemic protease inhibitor response in Nicotiana attenuata.JChemEcol Pearce G, Johnson S, Ryan CA (1993) Structure-activity of deleted and 27: 547–568

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