Generation of systemin signaling in tobacco by transformation with the tomato systemin receptor kinase gene

Abstract

The tomato systemin receptor, SR160, a plasma membrane-bound, leucine-rich repeat receptor kinase that signals systemic plant defense, and the brassinolide (BL) receptor, BRI1, that regulates developmental processes, have been shown recently to have identical amino acid sequences. We report herein that tobacco, a solanaceous species that does not express a systemin precursor gene nor responds to systemin, when transformed with the SR160 receptor gene, expresses the gene in suspension-cultured cells, evidenced by mRNA and protein analyses and photoaffinity-labeling experiments. Additionally, systemin induced an alkalinization response in the transgenic tobacco cells similar to that found in tomato cells, but not in WT cells. The gain in function in tobacco cells indicates that early steps of the systemin signaling pathway found in tomato are present in tobacco cells. A tomato line, cu-3, in which a mutation in the BRI1 gene has rendered the plant nonfunctional in BL signaling, exhibits a severely reduced response to systemin. In leaves of WT tomato plants, BL strongly and reversibly antagonized systemic signaling by systemin. The results suggest that the systemin-mediated systemic defense response may have evolved in some solanaceous species by co-opting the BRI1 receptor and associated components for defense signaling.

Wound-inducible defense genes are regulated systemically in several species of the Solanaceae family by the 18-aa polypeptide hormone systemin (1). Systemin is derived from a 200-aa precursor in response to wounding (2) and interacts with a plasma membrane receptor, SR160, to initiate an intracellular defense signaling cascade (3–5). Systemin has been identified so far only in members of the subtribe Solaneae of the Solanaceae family, including tomato, potato, black nightshade, and pepper (6), but not in tobacco, a member of the subtribe Nicotianae. SR160 protein and its cDNA were isolated, which identified the receptor as a cell surface, single-pass, leucine-rich repeat receptor kinase (4) with homology to the Arabidopsis brassinolide (BL) receptor, BRI1. The tomato BL receptor, tBRI1 (7), was recently isolated and shown to have an identical nucleotide sequence as SR160, indicating that the SR160/BRI1 receptor kinase in tomato has dual functions. However, unlike systemin signaling, BL signaling is ubiquitous in the plant kingdom (8), where it plays a fundamental role in growth and development (9). We present evidence here that the SR160 is a functionally active systemin receptor by expressing the tomato SR160 gene in tobacco, which does not express an endogenous prosystemin gene and does not respond to tomato systemin. In a tomato mutant line (cu-3) of a tomato species with a mutated, nonfunctional BRI1 receptor, systemin signaling is severely curtailed. The data support a dual functionality for the SR160/BRI1 receptor kinase in defense and development.

Materials and Methods

Plant Transformation. Leaf sections from 4-week-old tobacco plants grown in aseptic culture were transformed with the tomato SR160 cDNA obtained from Lycopersicon peruvianum suspension-cultured cells (4). The plant transformation vector pART27 was constructed containing the cauliflower mosaic virus 35S promoter ligated to the entire SR160 coding sequence of the cDNA in the sense orientation, from the methionine through the stop codon. The vector was used to transform tobacco leaf sections, mediated by Agrobacterium tumefaciens strain LBA4404 (Invitrogen) as described (10). Numerous shoots displaying kanamycin resistance on solid media were obtained, but only one of the shoots developed roots. All of the other transformants produced small leaves, but not roots. Gel blot analyses of leaf RNA from the rooted transformant confirmed that it strongly expressed the SR160 gene, whereas WT tobacco did not. The plant was grown to maturity and produced seeds, which segregated when grown on kanamycin. Leaf sections from the transformed plant were used to initiate calli and suspension cultures. Leaf sections were cultured on 2.0 mg/ml 2,4-d Murashige and Skoog (MS) solid media for 10 days, then transferred to 0.2 mg/ml 2,4-d MS solid media for 2 weeks. The resulting calli were placed in 50 ml of liquid media with 200 mg/liter kanamycin to initiate growth of suspension cultured cells, which were subcultured every 7–10 days.

Analysis of Tobacco Plants and Cells. RNA (10 μg) isolated from leaves of WT and transgenic plants by using TRIzol (Invitrogen) was subjected to electrophoresis, blotted to nylon, and probed with a 450-bp ³²P-labeled oligonucleotide derived from the SR160 cDNA as described (4). SR160 proteins in membranes from WT and transformed cells were subjected to 7.5% SDS/PAGE and analyzed for SR160 protein by protein blotting with a specific rabbit anti-SR160 serum and an alkaline phosphatase goat anti-rabbit secondary antibody (Bio-Rad) and Lumi-Phos WB (Pierce) to visualize the SR160 antibody.

SR160 was identified in the cell surface membranes of intact transformed tobacco suspension-cultured cells by using the photoaffinity reagent ¹²⁵I-azido-Cys-3, Ala-15-systemin using techniques and protocols previously used to identify the receptor in tomato plasma membranes (4). To 1 ml of each suspension culture, 2 × 10⁶ cpm of ¹²⁵I-azido-Cys-3, Ala-15-systemin (1 pmol) was added in the presence or absence of 200 pmol competing tomato systemin and incubated on an orbital shaker in the dark for 5 min. The cells were exposed to UVB irradiation, and membrane proteins were subjected to SDS/PAGE followed by exposure to x-ray film to detect radio-labeled SR160.

Alkalinization Assay. Suspension cells were maintained in Murashige and Skoog medium as described (4, 11), but excluding buffer, with the medium adjusted to pH 5.6 with KOH. Cultures were maintained by transferring 3 ml of cells to 45 ml of media every 7 days with shaking at 160 rpm. Tomato cells were used for alkalinization assays 4–7 days after transfer. One hour before assaying for alkalinating activity, 1-ml aliquots of cells were transferred into each well of 24-well cell-culture cluster plates and allowed to equilibrate while agitating on a rotating shaker at 160 rpm. Systemin, inactive systemin analog, or alanine-17 systemin (1–10 μl) were added to the cells, and the increase in pH of the medium was recorded after 15 min. The tobacco cells responded poorly when initially cultured, but with each transfer, the alkalinization response increased. The experiments recorded here were with cells that had undergone at least 10 transfers.

Tomato Bioassay. WT Lycopersicon pimpinellifolium and cu-3 mutant tomato plants (3–4 weeks old) were excised at the base of the stem and supplied with either water or 25 nM systemin through their cut stems for 1 h, followed by incubation under 200 μEm^–2·s^–1 of light at 28°C for 24 h. The plants were sectioned into upper, middle, and lower leaves (see Fig. 4A), and each section was analyzed for proteinase inhibitor content by radial immunodiffusion assays (12).

Fig. 4.

The tomato mutant cu-3 is defective in defense signaling. (A)AWT L. pimpinellifolium tomato plant (Left) and a cu-3 mutant plant (Right). (B) Intact WT and cu-3 mutant plants were exposed to methyl jasmonate vapors (MJ) as described in Materials and Methods. L, lower; M, middle; U, upper. Leaves were assayed for proteinase inhibitor II content by radial immunodiffusion. (C)WT L. pimpinellifolium plants and cu-3 mutant plants (both 3–4 weeks old) were excised at the base of the stem and supplied with either water or 25 nM systemin (Sys) through their cut stems as described in Materials and Methods. The leaves from the upper third of the plant (U), leaves from the middle (M), and leaves from the lower section (L) of the plants were pooled and assayed for proteinase inhibitor II as in B.

The induction of proteinase inhibitor protein in response to methyl jasmonate in intact WT and cu-3 mutant tomato plants was carried out as described (13). After exposure to methyl jasmonate vapors, plants were subsequently incubated under 200 μEm^–2·s^–1 of light at 28°C for 24 h, and proteinase inhibitor protein content in leaves was determined as described above.

In competition experiments with systemin and BL (CIDtech Research, Cambridge, Ontario, Canada), BL was first supplied to 14-day-old tomato plants (Lycopersicon esculentum) through their cut stems for 45 min, followed by solutions containing the indicated concentration of systemin for 45 min. The plants were incubated under constant light (200 μEm^–2·s^–1) at 28°C for 24 h and assayed for proteinase inhibitor content as above.

Results and Discussion

Transformation of tobacco leaves with the vector containing the tomato SR160 cDNA under control of the cauliflower mosaic virus 35S promoter resulted in numerous kanamycin-resistant tobacco plantlets when cultured in solid medium. However, among the plantlets, only one developed roots. The reason for the absence of root development by the cultured shoots is not known, but it is possible that overexpression of the receptor in tobacco may be interfering with normal root growth and development, perhaps related to the dual nature of the receptor in both systemin and BL signaling. The single transformant that did develop roots grew normally and set seeds.

To assess the expression of the SR160 transgene in the tobacco transformant, suspension cultures were grown from leaf tissues, and the cells were assessed for the synthesis of SR160 mRNA and protein. Suspension-cultured cells prepared from the transformed tobacco plant strongly expressed the SR160 gene, as evidenced by RNA blot analyses (Fig. 1A) and protein blot analyses (Fig. 1B). Neither SR160 mRNA nor protein were detected in suspension-cultured cells derived from WT tobacco plants.

Fig. 1.

Analysis of the expression of the systemin receptor SR160 in WT and transgenic tobacco cells (Nicotiana tabacum) overexpressing the SR160 cDNA. (A) RNA blot analysis of the SR160 mRNA in WT and transformed (TR) tobacco leaf tissue using an SR160-specific oligonucleotide probe. (B) Protein blot analysis of SR160 protein in WT and transformed (TR) suspension-cultured cells using an SR160 specific antibody.

To determine whether the tomato SR160 protein was present in cell-surface membranes of the suspension-cultured transgenic tobacco cells, as found previously in tomato cells, the transgenic tobacco cells and WT untransformed tobacco suspension-cultured cells were exposed to the photoaffininty reagent ¹²⁵I-azido-Cys-3,Ala-15-systemin, and the membrane proteins were analyzed by SDS/PAGE for receptor labeling. This reagent had previously been shown to interact with a single, 160-kDa plasma membrane protein on the surface of tomato suspension cultured cells (4). As with tomato cells, a single 160-kDa protein was labeled on the cell surface membranes of transgenic tobacco. The reagent did not label suspension-cultured cells of WT tobacco (Fig. 2). The labeling of the transgenic cells was blocked by a competing concentration of systemin that was known to compete with the photoaffinity label in tomato suspension-cultured cells (4). These cumulative results indicated that the tomato SR160 gene was expressed in the transgenic tobacco cells, resulting in the synthesis of SR160 mRNA and protein, and that the protein was targeted to the plasma membrane where it retained its systemin-binding capability.

Fig. 2.

Identification of SR160 on the cell surface of WT and transformed (TR) tobacco (Tob) and tomato (Tom) cells by photoaffinity labeling with ¹²⁵I-azido-Cys-3, Ala-15-systemin. To 1 ml of each suspension culture, 2 × 10⁶ cpm ¹²⁵I-azido-Cys-3, Ala-15-systemin (1 pmol) was added in the absence (–Sys) or presence (+Sys) of 200 pmol tomato systemin and photo-labeled as described in Materials and Methods. Membrane proteins from labeled cells were subjected to SDS/PAGE analysis followed by exposure to x-ray film to detect radio-labeled SR160.

Because the tomato SR160 receptor protein in tobacco could recognize systemin, the transgenic tobacco cells were assessed for their ability to respond to the systemin–SR160 interaction by activating the intracellular mechanism leading to the characteristic alkalinization that is part of the defense-signaling pathway of tomato cells in response to systemin (3, 11). Systemin induced a typical alkalinization response when added to the transgenic tobacco cells (Fig. 3A), a response not found with WT tobacco cells (14). A dose–response analysis of the systemin-induced alkalinization of the transgenic tobacco cells (Fig. 3B) determined that tomato systemin has an EC₅₀ ≈0.8 nM in the transgenic tobacco, identical to the EC₅₀ of systemin that is found in the alkalinization response of tomato cells (3, 4).

Fig. 3.

Systemin induces an alkalinization response in suspension-cultured tobacco cells overexpressing SR160. (A) The change in extracellular pH of WT and transgenic (TR) tobacco cells overexpressing SR160 in response to 250 nM systemin (SYS). (B) Concentration dependence of the induced alkalinization of cells by systemin (SYS) and the inactive Ala-17-systemin (A17). Changes in pH were recorded 15 min after addition of the peptides. Data points are representative of three independent experiments and are the mean of two samples. Bars indicate standard deviation.

To further investigate the dual functionality of the tomato SR160/BRI1 receptor, a tomato (L. pimpinellifolium) mutant, cu-3, that is nonfunctional in BR signaling (7) (Fig. 4A) was assessed for its ability to respond to both methyl jasmonate and systemin, compared with WT plants. The plants were first exposed to methyl jasmonate vapors to establish that the downstream signaling pathway was intact. The cu-3 plants synthesized inhibitor protein in all leaves in response to methyl jasmonate, although the response was much lower than in leaves of WT plants (Fig. 4B). This finding indicated that the signaling pathway downstream from the receptor was functional in the mutant, but at a lower level than in the WT plants. Cu-3 plants did not exhibit an induction of proteinase inhibitor by systemin in the lower and middle leaves (Fig. 4C), but did exhibit a moderate increase in the upper apical leaves. This induction was much less than found in WT plants treated with either methyl jasmonate or systemin. This finding indicated that in the apical region of the plant an independent mechanism for wound activation of proteinase inhibitors may be present.

The lower response of the cu-3 plants to methyl jasmonate than WT plants suggested that a functional SR160/BRI1 receptor kinase could not be induced by methyl jasmonate, as it is in WT plants (4). This finding suggests that the reduced effects of methyl jasmonate in cu-3 plants may be caused by an impaired amplification of systemin signaling that occurs through the systemin receptor (5).

In a previous report, BL did not compete with systemin when assayed in the alkalinization assay with tomato suspension-cultured cells (3). However, in experiments using young, excised tomato plants, we have found that supplying the plants with BL, followed by systemin, strongly antagonized systemin-induced inhibitor I protein synthesis (Fig. 5A). An IC₅₀ of <0.5 μM BL was calculated for the inhibition of 2.5 nM systemin. This inhibitory effect appears to be competitive, because the inhibition can be overcome by increasing concentrations of systemin (Fig. 5B). The lack of BL competition for systemin in the alkalinization assay with cultured cells may reflect the lack of some component(s) of the BL signaling complex that is present in intact plants. The competition between systemin and BL suggests that signaling through the SR160/BRI1 receptor kinase in intact tomato plants may reflect the levels of systemin or BL that are present in tissues in response to environmental and developmental queues. The functional antagonism between ligands observed here is reminiscent of the oxytocin-progesterone antagonism that occurs with the seven-span oxytocin GPC receptor reported in animals (15). The Toll receptor in Drosophila is another example of a leucine-rich repeat receptor with two diverse functions (16, 17), in the development of the dorsal-ventral axis, and later in innate immunity.

Fig. 5.

BL competitively inhibits systemin-induced proteinase inhibitor synthesis in leaves of excised tomato plants. (A) Fourteen-day-old excised tomato plants (L. esculentum) were supplied with solutions of BL through their cut stems for 45 min, followed by solutions containing 2.5 nM systemin (SYS) for 45 min. Proteinase inhibitor concentrations were determined 24 h later as described in Materials and Methods. (B) BL at 10 μM was supplied to young excised tomato plants for 45 min followed by the indicated concentration of systemin (SYS) for 45 min. The plants were incubated in the same conditions of light and temperature as above for 24 h and assayed for proteinase inhibitor protein as in Fig. 4.

The alkalinization response of transgenic tobacco cells expressing the tomato SR160 gene, when challenged with systemin, implies that an intracellular signaling component(s) of tobacco can interact with SR160 to facilitate signaling. The ancient BL signaling pathway may have had, and may still have, a defensive component that allowed the co-opting of the pathway by systemin as an evolutionary adaptation, an adaptation that is now present in several species of the subtribe Solaneae. An understanding of the signaling components associated with the dual functionality of the BRI1/SR160 receptor kinase should reveal insights into the evolution of this dual-function receptor and provide opportunities for enhancing natural defense in crop plants.