Suramin inhibits initiation of defense signaling by systemin, chitosan, and a beta-glucan elicitor in suspension-cultured Lycopersicon peruvianum cells

Abstract

Systemin-mediated defense signaling in tomato (Lycopersicon esculentum) plants is analogous to the cytokine-mediated inflammatory response in animals. Herein, we report that the initiation of defense signaling in suspension-cultured cells of Lycopersicon peruvianum by the peptide systemin, as well as by chitosan and beta-glucan elicitor from Phytophtora megasperma, is inhibited by the polysulfonated naphtylurea compound suramin, a known inhibitor of cytokine and growth factor receptor interactions in animal cells. Using a radioreceptor assay, we show that suramin interfered with the binding of the systemin analog (125)I-Tyr-2, Ala-15-systemin to the systemin receptor with an IC(50) of 160 microM. Additionally, labeling of the systemin receptor with a photoaffinity analog of systemin was inhibited in the presence of suramin. Receptor-mediated tyrosine phosphorylation of a 48-kDa mitogen-activated protein kinase and alkalinization of the medium of suspension-cultured cells in response to systemin and carbohydrate elicitors were also inhibited by suramin. The inhibition of medium alkalinization by suramin was reversible in the presence of high concentrations of systemin and carbohydrate elicitors. Calyculin A and erythrosin B, intracellular inhibitors of phosphatases and plasma membrane proton ATPases, respectively, both induce medium alkalinization, but neither response was inhibited by suramin. The polysulfonated compound heparin did not inhibit systemin-induced medium alkalinization. NF 007, a suramin derivative, induced medium alkalinization, indicating that neither NF 007 nor heparin interact with elicitor receptors like suramin. The data indicate that cell-surface receptors in plants show some common structural features with animal cytokine and growth factor receptors that can interact with suramin to interfere with ligand binding.

Figure 1

Systemin-, chitosan-, and pmg elicitor-induced medium alkalinization is inhibited by suramin. Cells of L. peruvianum were left untreated (●, − SUR) or treated for 10 min with 700 μM suramin (○, + SUR). Subsequently, systemin (A), chitosan (B), or pmg elicitor (C) was supplied at the concentrations indicated. The pH of the medium was measured 20 min after addition of elicitors. The increase in medium pH (Δ pH) is indicated as the difference between the pH at time 0 and at 20 min.

Figure 2

Inhibition by suramin of medium alkalinization in response to systemin, chitosan, and pmg elicitor is concentration dependent. Cells of L. peruvianum were treated for 10 min with suramin at the concentrations indicated. The cells were then supplied with 1 nM systemin (○), 125 ng/ml chitosan (●), or 50 μg/ml pmg elicitor (×), and the pH change of the growth medium was determined after 7 min (systemin), 10 min (chitosan), or 20 min (pmg elicitor). The pH change is expressed as percentage of inhibition of medium alkalinization in the absence of suramin.

Figure 3

Suramin blocks binding of ¹²⁵I-Tyr-2,Ala-15-systemin to its cell-surface binding site. Cells of L. peruvianum were treated for ≤1 min with suramin at the concentrations indicated. The cells were then supplied with 1 nM ¹²⁵I-Tyr-2,Ala-15-systemin with or without a 200-fold excess of unlabeled systemin. After 7 min, the cells were washed, and the specific binding of systemin to the cells was determined by subtracting total binding from unspecific binding (●). The data are expressed as percentage of inhibition of specific binding in the absence of suramin. For comparison, the effect of suramin on medium alkalinization in response to systemin is shown (○).

Figure 4

Suramin interferes with photoaffinity labeling of the systemin receptor. ¹²⁵I-N-(4-[p-azidosalicylamido]butyl)-3′(2′-Cys-3,Ala-15-systemindithiol)propionamide was incubated with suspension-cultured cells in the absence (lane 1) or presence (lane 2) of 150 pmol unlabeled systemin or in the presence of 1 mM suramin (lane 3). After photoactivation of the crosslinker, microsomal membranes were prepared, and membrane proteins were separated by SDS/PAGE. The radiolabeled receptor protein was visualized by phosphorimaging. Numbers and arrows at left indicate the position and molecular mass (in kDa) of protein standards.

Figure 5

Systemin, chitosan, and pmg elicitor activation of 48-kDa MBP kinase is inhibited by suramin. (A) MBP kinase activity. Cells of L. peruvianum were pretreated for 5 min with 1 mM suramin (SUR +) or left untreated (SUR −). They were then supplied with 3.3 nM systemin (Sys), 0.5 μg/ml chitosan (Chi), 50 μg/ml pmg elicitor (Pmg), or water (C). At the times indicated after addition of the elicitors, cells were frozen, extracted, and assayed for MBP kinase activity with an in-gel kinase assay. (B) Immunocomplex kinase assay. Phosphotyrosine-containing proteins in the extracts of samples (20 min) shown in A were complexed with a monoclonal phosphotyrosine-specific antibody coupled to Sepharose 4B and washed. Immunocomplexes were then analyzed for MBP kinase activity with an in-gel kinase assay.

Figure 6

Effect of suramin on medium alkalinization induced by systemin, calyculin A, and erythrosin B. Cells of L. peruvianum were either left untreated (Sur −) or treated with 1,000 μM suramin (Sur +) for 5 min. Thereafter, 3.3 nM systemin (Sys), 2 μM calyculin A (Cal), or 20 μM erythrosin B (EB) was added to the cells. The pH of the medium was measured 10 min after addition of elicitors, and the increase in medium pH (Δ pH) was determined as described in Fig. 2.

Figure 7

Analogies between animal and plant defense-signaling pathways. (A) Early signaling events of the tomato wound response (47). (B) Early signaling events of the animal inflammatory response (20). A, plasma membrane proton ATPase; ER, endoplasmic reticulum; IP₃, inositoltriphosphate; PLA₂, phospholipase A₂; PLC, phospholipase C; PM, plasma membrane; R, receptor; TNFα, tumor necrosis factor-α; V, vacuole.