A plasma membrane protein from Zea mays binds with the herbivore elicitor volicitin

Abstract

Volicitin (17-hydroxylinolenoyl-l-Gln) present in the regurgitant of Spodoptera exigua (beet armyworm caterpillars) activates the emission of volatile organic compounds (VOCs) when in contact with damaged Zea mays cv Delprim (maize) leaves. VOC emissions in turn serve as a signaling defense for the plant by attracting female parasitic wasps that prey on herbivore larvae. A tritiated form of volicitin was synthesized and shown to induce volatiles in the same fashion as the biological form. [(3)H]-l-volicitin rapidly, reversibly, and saturably bound to enriched plasma membrane fractions isolated from Z. mays leaves with an apparent K(d) of 1.3 nM and a Hill coefficient of 1.07. Analog studies showed that the l-Gln and hydroxy moieties of volicitin play an important role in binding. Treatment of plants with methyl jasmonate (MeJA) increased the total binding of [(3)H]-l-volicitin to the enriched plasma membrane more than threefold, suggesting that MeJA activates transcription of the gene encoding the binding protein. S. exigua feeding also increased total binding fourfold. Cycloheximide pretreatment of plants significantly decreased binding of radiolabeled volicitin to the enriched plasma membrane. These data provide the first experimental evidence that initiation of plant defenses in response to herbivore damage can be mediated by a binding protein-ligand interaction.

Figure 1.

Verification of Synthetic Volicitin Activity Using a Z. mays Bioassay. Release of volatiles collected for 2 h from six Z. mays seedlings that had been treated with 300 pmol per plant of l-volicitin, d-volicitin, 17-hydroxylinolenic acid, l-Gln, and linolenoyl-l-Gln, with concentrations ranging from 50 μg/μL to 100 μg/μL plus buffer for a final volume of 500 μL. The combined amount in nanograms of caryophyllene, α-trans-bergamontene, (E)-β-farnesene, (E)-nerolidol, and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene was used to calculate the relative release to that of seedlings treated with 15 μL of BAW oral secretions plus 485 μL buffer, which is equivalent to ∼300 pmol natural volicitin. Data labeled with the same letter do not differ significantly (ANOVA, P < 0.05).

Figure 2.

Chemical Structure of Volicitin and Analogs Assayed. (A) l-volicitin. (B) d-volicitin. (C) Linolenoyl-l-Gln. (D) 17-hydroxylinolenic acid. (E) l-Gln.

Figure 3.

Time Course of [³H]-l-Volicitin Binding to Z. mays Enriched Plasma Membranes. Binding of [³H]-l-volicitin (10 nM) in the absence (closed circle, total binding) or presence (open circle, nonspecific binding) of a 100-fold excess of unlabeled l-volicitin over time. At the indicated times, the membranes were collected, washed, and analyzed for radioactivity. Error bars indicate standard error (n = 6).

Figure 4.

Competition of Unlabeled Volicitin and Volicitin Analogs with [³H]-l-Volicitin for Binding Sites on the Z. mays Enriched Plasma Membranes. (A) Competition of [³H]-l-volicitin binding to enriched plasma membranes with unlabeled l-volicitin, d-volicitin, linolenoyl-l-Gln, 17-hydroxylinolenic acid (17-hydroxy), and l-Gln. The competing analogs were added at a 100-fold molar excess (1 μM) over [³H]-l-volicitin (10 nM). Data labeled with the same letter do not differ significantly (ANOVA, P < 0.05). (B) Competition analysis of [³H]-l-volicitin binding by l-volicitin, d-volicitin, and linolenoyl-l-Gln were determined by treating the enriched plasma membrane preparations with the indicated concentrations of unlabeled analog and by calculating the percentage of specific binding as a ratio of specific binding at the indicated concentration to maximal specific binding found in the presence of 100-fold excess of unlabeled volicitin. Closed circles, L-volicitin; open circles, D-volicitin; triangles, linolenoyl-L-Gln. Error bars indicate standard error (n = 6).

Figure 5.

Saturation Analysis of l-Volicitin and Linolenoyl-l-Gln Induction of VOCs in Z. mays Seedlings. Maximum release of volatiles collected for 2 h from six Z. mays seedlings that had been treated with the indicated concentration of either l-volicitin (closed circles) or linolenoyl-l-Gln (open circles). The combined amount in nanograms of caryophyllene, α-trans-bergamontene, (E)-β-farnesene, (E)-nerolidol, and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene was used to calculate the release of seedlings treated with 15 μL of test solution. Error bars indicate standard error (n = 6).

Figure 6.

Saturation Analysis of [³H]-l-Volicitin Binding to Z. mays Enriched Plasma Membranes. (A) Enriched plasma membrane preparations were treated with increasing concentrations of [³H]-l-volicitin. The specific binding (total binding minus nonspecific binding) is shown. The specific activity of [³H]-l-volicitin was ∼1.2 Ci/mmol (140 dpm/fmol). Error bars indicate standard error (n = 6). (B) Scatchard analysis of [³H]-l-volicitin binding to enriched plasma membranes from data shown in (A). The K_d was calculated from the negative inverse of the slope and B_max from the x-intercept. (C) Hill analysis of [³H]-l-volicitin binding to enriched plasma membranes derived from the data shown in (A). The slope of the plot is the Hill coefficient (n_Hill).

Figure 7.

Reversibility of [³H]-l-Volicitin Binding to Z. mays Enriched Plasma Membranes. Two sets of cells were treated with saturating levels of [³H]-l-volicitin (10 nM), and the total radioactivity associated with the enriched plasma membrane was determined at the indicated time. A 100-fold excess (1 μM) of unlabeled volicitin was added to one set of enriched plasma membrane preparations 4 min after the addition of [³H]-l-volicitin. The total radioactivity associated with both sets of enriched plasma membranes was determined at the indicated times. Error bars indicate standard error (n = 6).

Figure 8.

Assay pH Influences [³H]-l-Volicitin–Plasma Membrane Binding. [³H]-l-Volicitin (10 nM) filter binding and slot-blot assays for plasma membrane enriched fractions adjusted to the selected pH. Slot-blot insets display [³H]-l-volicitin binding to enriched plasma membranes bound to nitrocellulose and analyzed by autoradiography. Data labeled with the same letter do not differ significantly (ANOVA, P < 0.05).

Figure 9.

Induction by MeJA, BAW, and Mechanical Wounding on [³H]-l-Volicitin–Plasma Membrane Binding. Saturating levels of [³H]-l-volicitin (10 nM) were used to determine the total level of binding at the indicated times after treatment with MeJA, BAW, or razor blade. The total binding is shown in fmol·μg protein⁻¹. Error bars indicate standard error (n = 6).

Figure 10.

Protein Requirement for [³H]-l-Volicitin–Plasma Membrane Binding. [³H]-l-volicitin (10 nM) filter binding and slot-blot assays with indicated plant and plasma membrane fraction treatments. Slot-blot insets display [³H]-l-volicitin (1.6 × 10⁶ cpm·mL⁻¹) binding to enriched plasma membranes bound to nitrocellulose and analyzed by autoradiography. Asterisks indicate the lower fraction (L₁) containing a non-plasma membrane enriched fraction. Data labeled with the same letter do not differ significantly (ANOVA, P < 0.05).