Generation of systemin signaling in tobacco by transformation with the tomato systemin receptor kinase gene Justin M. Scheer*, Gregory Pearce, and Clarence A. Ryan† Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340 Contributed by Clarence A. Ryan, May 14, 2003 The tomato systemin receptor, SR160, a plasma membrane-bound, tomato SR160 cDNA obtained from Lycopersicon peruvianum leucine-rich repeat receptor kinase that signals systemic plant suspension-cultured cells (4). The plant transformation vector defense, and the brassinolide (BL) receptor, BRI1, that regulates pART27 was constructed containing the cauliflower mosaic developmental processes, have been shown recently to have virus 35S promoter ligated to the entire SR160 coding sequence identical amino acid sequences. We report herein that tobacco, a of the cDNA in the sense orientation, from the methionine solanaceous species that does not express a systemin precursor through the stop codon. The vector was used to transform gene nor responds to systemin, when transformed with the SR160 tobacco leaf sections, mediated by Agrobacterium tumefaciens receptor gene, expresses the gene in suspension-cultured cells, strain LBA4404 (Invitrogen) as described (10). Numerous shoots evidenced by mRNA and protein analyses and photoaffinity-label- displaying kanamycin resistance on solid media were obtained, ing experiments. Additionally, systemin induced an alkalinization but only one of the shoots developed roots. All of the other response in the transgenic tobacco cells similar to that found in transformants produced small leaves, but not roots. Gel blot tomato cells, but not in WT cells. The gain in function in tobacco analyses of leaf RNA from the rooted transformant confirmed cells indicates that early steps of the systemin signaling pathway that it strongly expressed the SR160 gene, whereas WT tobacco found in tomato are present in tobacco cells. A tomato line, cu-3, did not. The plant was grown to maturity and produced seeds, in which a mutation in the BRI1 gene has rendered the plant which segregated when grown on kanamycin. Leaf sections from nonfunctional in BL signaling, exhibits a severely reduced response the transformed plant were used to initiate calli and suspension to systemin. In leaves of WT tomato plants, BL strongly and cultures. Leaf sections were cultured on 2.0 mg͞ml 2,4-D Mu- reversibly antagonized systemic signaling by systemin. The results rashige and Skoog (MS) solid media for 10 days, then transferred suggest that the systemin-mediated systemic defense response to 0.2 mg͞ml 2,4-D MS solid media for 2 weeks. The resulting may have evolved in some solanaceous species by co-opting the calli were placed in 50 ml of liquid media with 200 mg͞liter BRI1 receptor and associated components for defense signaling. kanamycin to initiate growth of suspension cultured cells, which were subcultured every 7–10 days. prosystemin ͉ SR160 ͉ BRI1 ͉ plant defense ͉ plant development Analysis of Tobacco Plants and Cells. RNA (10 ␮g) isolated from leaves of WT and transgenic plants by using TRIzol (Invitrogen) ound-inducible defense genes are regulated systemically was subjected to electrophoresis, blotted to nylon, and probed in several species of the Solanaceae family by the 18-aa W with a 450-bp 32P-labeled oligonucleotide derived from the polypeptide hormone systemin (1). Systemin is derived from a SR160 cDNA as described (4). SR160 proteins in membranes 200-aa precursor in response to wounding (2) and interacts with from WT and transformed cells were subjected to 7.5% SDS͞ a plasma membrane receptor, SR160, to initiate an intracellular PAGE and analyzed for SR160 protein by protein blotting with defense signaling cascade (3–5). Systemin has been identified so a specific rabbit anti-SR160 serum and an alkaline phosphatase far only in members of the subtribe Solaneae of the Solanaceae goat anti-rabbit secondary antibody (Bio-Rad) and Lumi-Phos family, including tomato, potato, black nightshade, and pepper WB (Pierce) to visualize the SR160 antibody. (6), but not in tobacco, a member of the subtribe Nicotianae. SR160 was identified in the cell surface membranes of intact SR160 protein and its cDNA were isolated, which identified the transformed tobacco suspension-cultured cells by using the receptor as a cell surface, single-pass, leucine-rich repeat recep- photoaffinity reagent 125I-azido-Cys-3, Ala-15-systemin using tor kinase (4) with homology to the Arabidopsis brassinolide techniques and protocols previously used to identify the receptor (BL) receptor, BRI1. The tomato BL receptor, tBRI1 (7), was in tomato plasma membranes (4). To 1 ml of each suspension recently isolated and shown to have an identical nucleotide ϫ 6 125 ͞ culture, 2 10 cpm of I-azido-Cys-3, Ala-15-systemin (1 sequence as SR160, indicating that the SR160 BRI1 receptor pmol) was added in the presence or absence of 200 pmol kinase in tomato has dual functions. However, unlike systemin competing tomato systemin and incubated on an orbital shaker signaling, BL signaling is ubiquitous in the plant kingdom (8), in the dark for 5 min. The cells were exposed to UVB irradiation, where it plays a fundamental role in growth and development and membrane proteins were subjected to SDS͞PAGE followed (9). We present evidence here that the SR160 is a functionally by exposure to x-ray film to detect radio-labeled SR160. active systemin receptor by expressing the tomato SR160 gene in tobacco, which does not express an endogenous prosystemin Alkalinization Assay. Suspension cells were maintained in Mu- gene and does not respond to tomato systemin. In a tomato rashige and Skoog medium as described (4, 11), but excluding mutant line (cu-3) of a tomato species with a mutated, nonfunc- buffer, with the medium adjusted to pH 5.6 with KOH. Cultures tional BRI1 receptor, systemin signaling is severely curtailed. were maintained by transferring 3 ml of cells to 45 ml of media The data support a dual functionality for the SR160͞BRI1 receptor kinase in defense and development. Abbreviation: BL, brassinolide. Materials and Methods *Present address: Sunesis Pharmaceuticals, Inc., 341 Oyster Point Boulevard, South San Plant Transformation. Leaf sections from 4-week-old tobacco Francisco, CA 94080. plants grown in aseptic culture were transformed with the †To whom correspondence should be addressed. E-mail: [email protected]. 10114–10117 ͉ PNAS ͉ August 19, 2003 ͉ vol. 100 ͉ no. 17 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1432910100 Downloaded by guest on September 28, 2021 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 ␮l) 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 ␮EmϪ2⅐sϪ1 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). 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 ␮EmϪ2⅐sϪ1 of light at 28°C for 24 h, and proteinase inhibitor protein content in leaves was determined as de- scribed above. Fig. 1. Analysis of the expression of the systemin receptor SR160 in WT and In competition experiments with systemin and BL (CIDtech transgenic tobacco cells (Nicotiana tabacum) overexpressing the SR160 cDNA. Research, Cambridge, Ontario, Canada), BL was first supplied (A) RNA blot analysis of the SR160 mRNA in WT and transformed (TR) tobacco to 14-day-old tomato plants (Lycopersicon esculentum) through leaf tissue using an SR160-specific oligonucleotide probe. (B) Protein blot their cut stems for 45 min, followed by solutions containing the analysis of SR160 protein in WT and transformed (TR) suspension-cultured cells indicated concentration of systemin for 45 min. The plants were using an SR160 specific antibody. incubated under constant light (200 ␮EmϪ2⅐sϪ1)at28°C for 24 h and assayed for proteinase inhibitor content as above. The reagent did not label suspension-cultured cells of WT Results and Discussion tobacco (Fig. 2). The labeling of the transgenic cells was blocked PLANT BIOLOGY Transformation of tobacco leaves with the vector containing the by a competing concentration of systemin that was known to tomato SR160 cDNA under control of the cauliflower mosaic compete with the photoaffinity label in tomato suspension- virus 35S promoter resulted in numerous kanamycin-resistant cultured cells (4). These cumulative results indicated that the tobacco plantlets when cultured in solid medium. However, tomato SR160 gene was expressed in the transgenic tobacco among the plantlets, only one developed roots. The reason for cells, resulting in the synthesis of SR160 mRNA and protein, and the absence of root development by the cultured shoots is not that the protein was targeted to the plasma membrane where it known, but it is possible that overexpression of the receptor in retained its systemin-binding capability. tobacco may be interfering with normal root growth and devel- Because the tomato SR160 receptor protein in tobacco opment, perhaps related to the dual nature of the receptor in could recognize systemin, the transgenic tobacco cells were both systemin and BL signaling.