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. 2001 May;13(5):1079-93.
doi: 10.1105/tpc.13.5.1079.

A harpin binding site in tobacco plasma membranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activated protein kinase activity

J Lee  1 , D F Klessig, T Nürnberger
Affiliations

A harpin binding site in tobacco plasma membranes mediates activation of the pathogenesis-related gene HIN1 independent of extracellular calcium but dependent on mitogen-activated protein kinase activity

J Lee et al. Plant Cell. 2001 May.

Abstract

Harpin from the bean halo-blight pathogen Pseudomonas syringae pv phaseolicola (harpin(Psph)) elicits the hypersensitive response and the accumulation of pathogenesis-related gene transcripts in the nonhost plant tobacco. Here, we report the characterization of a nonproteinaceous binding site for harpin(Psph) in tobacco plasma membranes, which is assumed to mediate the activation of plant defense responses in a receptor-like manner. Binding of 125I-harpin(Psph) to tobacco microsomal membranes (dissociation constant = 425 nM) and protoplasts (dissociation constant = 380 nM) was specific, reversible, and saturable. A close correlation was found between the abilities of harpin(Psph) fragments to elicit the transcript accumulation of the pathogenesis-related tobacco gene HIN1 and to compete for binding of 125I-harpin(Psph) to its binding site. Another elicitor of the hypersensitive response and HIN1 induction in tobacco, the Phytophthora megasperma-derived beta-elicitin beta-megaspermin, failed to bind to the putative harpin(Psph) receptor. In contrast to activation by beta-megaspermin, harpin(Psph)-induced activation of the 48-kD salicylic acid-responsive mitogen-activated protein kinase (MAPK) and HIN1 transcript accumulation were independent of extracellular calcium. Moreover, use of the MAPK kinase inhibitor U0126 revealed that MAPK activity was essential for pathogenesis-related gene expression in harpin(Psph)-treated tobacco cells. Thus, a receptor-mediated MAPK-dependent signaling pathway may mediate the activation of plant defense responses induced by harpin(Psph).

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Figures

Figure 1.
Figure 1.
HarpinPsph-Induced Hypersensitive Cell Death and PR Gene Expression in Tobacco. HarpinPsph (1 μM) was infiltrated into tobacco leaves or added to suspension cultured tobacco cells. (A) Tobacco leaf treated with 5 mM Mes buffer, pH 5.5 (1), or harpinPsph (2) 2 days after infiltration. (B) Total RNA prepared from tobacco cells treated for 3 hr with buffer (−) or harpinPsph (+) was used as a template in RT-PCR assays with DNA primers derived from the tobacco genes indicated (see text). The tobacco gene encoding EF1α served as an internal control. PZ, chitinase/lysozyme. (C) Kinetics of HIN1 expression in tobacco cells treated with buffer or harpinPsph. Total RNA from tobacco cells was prepared at the times after infiltration indicated and used in RT-PCR to simultaneously amplify HIN1 and EF1α transcripts. (D) HIN1 expression in tobacco cells or leaves treated for 3 hr with buffer (−) or harpinPsph (+). RT-PCR analysis was performed as described in (C).
Figure 2.
Figure 2.
Binding of 125I-HarpinPsph to Tobacco Membranes. Data points represent the average of triplicate experiments. Data points representing nonspecific binding are the average of duplicate experiments. (A) Time course of binding of 50 nM 125I-harpinPsph (1:10 dilution with unlabeled harpinPsph) to tobacco microsomal membranes added at time 0. Membrane-bound radioligand was determined at the times indicated. Squares show the amount of radioligand that was bound specifically by the 125I-harpinPsph binding site. Nonspecific binding is indicated by triangles. Specific binding was obtained by subtracting nonspecific binding from total binding. Nonspecific binding was determined in the presence of 5 μM unlabeled harpinPsph. (B) Time course of displacement of 125I-harpinPsph. 125I-harpinPsph (50 nM; 1:10 dilution with unlabeled harpinPsph) was incubated with tobacco microsomal membranes for the times indicated. To initiate the displacement of 125I-harpinPsph, a 100-fold molar excess of unlabeled harpinPsph (open squares) was added 45 min (arrow) after the radioligand. Specific binding (closed squares) and nonspecific binding (triangles) are indicated. (C) Saturability of 125I-harpinPsph binding to tobacco microsomal membranes. Specific binding (closed squares) and nonspecific binding (open squares) were determined in the presence of a 100-fold molar excess of unlabeled harpinPsph. (D) Scatchard plot and Hill plot of the binding data shown in (C). The binding constant (KD) and the Hill coefficient (n) were determined according to Hulme and Birdsall (1990). (E) Saturability of 125I-harpinPsph binding to tobacco protoplasts. Graphs for specific binding (squares) and nonspecific binding (triangles) are shown. The inset shows a Scatchard plot of the binding data. The dissociation constant (KD) was determined according to Hulme and Birdsall (1990). (F) Competitive inhibition of 125I-harpinPsph (100 nM) binding to tobacco microsomal membranes by increasing concentrations of harpinPsph or β-megaspermin (β-MS). One hundred percent specific binding corresponded to the binding detected in the absence of competitor (1,600,000 cpm), whereas 0% specific binding corresponded to the binding detected in the presence of a 100-fold molar excess of competitor (10 μM) (nonspecific binding, 374,000 cpm). Error bars indicate standard error.
Figure 3.
Figure 3.
Structure/Activity Relationship of HarpinPsph and HarpinPsph Deletion Derivatives Expressed in E. coli. (A) cDNAs encoding harpinPsph (fragment I) and deletion derivatives (fragments II to VII) fused to a His10-encoding tag were expressed in E. coli. Expression products were purified to apparent homogeneity on nickel–nitrilotriacetic acid agarose and tested for their ability to induce HIN1 expression or the HR in tobacco cells at a concentration of 1 μM. Total RNA was prepared from tobacco cells treated with harpinPsph for 3 hr, and HIN1 expression was monitored by RT-PCR as described in the legend to Figure 1C. The white bar (fragment VIII) denotes the overlapping region of two active derivatives (fragments III and V) and defines the smallest fragment deduced to harbor elicitor activity. +, full activity relative to that of the full-length expression product; −, no detectable activity. (B) Competitive inhibition of 125I-harpinPsph (100 nM) binding to tobacco microsomal membranes by recombinant deletion derivatives shown in (A) and harpinPss from P. s. syringae. Increasing concentrations of fragment I (closed squares), fragment III (closed triangles), harpinPss (closed inverted triangles), fragment VI (open circles), or fragment VII (open diamonds) were used as competitors of binding of 100 nM 125I-harpinPsph. The graphs show the amount of specific binding as described in the legend to Figure 2A. One hundred percent specific binding corresponded to the binding detected in the absence of competitor (1,850,000 cpm), whereas 0% specific binding corresponded to the binding detected in the presence of a 100-fold molar excess of competitor (10 μM) (nonspecific binding, 525,000 cpm). Each data point represents the average of duplicate experiments.
Figure 4.
Figure 4.
HarpinPsph-Induced HIN1 Expression Is Independent of Extracellular Calcium. Effects of Ca2+ chelator and Ca2+ channel inhibitors on elicitor-induced HIN1 expression in tobacco cells. Tobacco cells were treated for 1.5 hr with buffer (C), 1 μM harpinPsph (H), or 50 nM β-megaspermin (β-MS) in the absence or presence of BAPTA (8 mM), LaCl3 (0.25 mM; La3+), or GdCl3 (0.25 mM; Gd3+). MgSO4 (20 mM) was included in the buffer together with BAPTA to prevent membrane destabilization due to Ca2+ depletion. Total RNA prepared from elicited tobacco cells was analyzed by RT-PCR as described in the legend to Figure 1C.
Figure 5.
Figure 5.
Activation of the SIPK in Tobacco Cells Treated with HarpinPsph. Tobacco cells treated with 1 μM harpinPsph were harvested at the times after infiltration indicated and used to prepare protein extracts. (A) Kinase activity determined by an in-gel kinase assay using MBP as the substrate. (B) Protein extracts analyzed by immunoblotting using an antiserum (Ab) recognizing the pTEpY motif of activated MAPK. (C) Protein extracts were prepared from tobacco cells treated with buffer (lane 1) or harpinPsph (lanes 2 and 3) for 5 min. For immunoprecipitation, the tobacco SIPK-specific antibody (Ab-p48N) (Zhang et al., 1998) was added alone (lanes 1 and 2) or together with the competitor peptide used for antibody production (lane 3). Kinase activity of immunoprecipitated material was assayed using MBP as the substrate. The phosphorylated MBP was visualized by autoradiography.
Figure 6.
Figure 6.
HarpinPsph-Induced SIPK Activation Is Independent of Extracellular Calcium. Protein extracts prepared from tobacco cells treated for 5 min with buffer, 1 μM harpinPsph, or 50 nM β-megaspermin (β-MS) in the absence (Control) or presence of the Ca2+ chelator BAPTA were analyzed for MAPK phosphorylation by immunoblotting using the anti-pTEpY-antiserum.
Figure 7.
Figure 7.
The MAPKK-Specific Inhibitor U0126 Compromises Both HarpinPsph-Induced SIPK Activation and PR Gene Expression in Tobacco. (A) Tobacco cells were treated for 3 hr with buffer (−) or 1 μM harpinPsph (+) in the absence (DMSO) or presence of 50 or 100 μM U0126, a MAPKK-specific inhibitor (Favata et al., 1998). The highest concentration of DMSO used was 0.2%. Protein extracts from elicited tobacco cells were analyzed for SIPK activity as described in the legend to Figure 5C. The expression of PR genes was analyzed by RT-PCR with total RNA and specific DNA primers for the indicated genes or, alternatively, by RNA gel blot analysis using total RNA and α-32P-dATP–labeled HSR203 cDNA. (B) Quantification of the inhibitory effect of U0126 on harpinPsph-induced HIN1 expression. RT-PCR products obtained from three independent elicitation experiments as described in (A) were separated electrophoretically, blotted onto nylon membranes, and probed with α-32P-dATP–labeled HIN1 cDNA. The signal intensity was quantified by phosphorimaging and normalized to 100% for the maximum induction in each experiment. Buffer treatment (blank bars); harpinPsph treatment (shaded bars). Error bars indicate ±se.
Figure 8.
Figure 8.
Hypothetical Model for HarpinPsph-Induced Signal Transduction in Tobacco. Activation by harpinPsph of pathogen defense responses in tobacco may be mediated by receptor-mediated membrane insertion (1), by direct insertion of harpinPsph into membranes, or by a specific (non)proteinaceous receptor (2). Alternatively, membrane insertion and receptor-mediated recognition of harpinPsph may occur independently, with either or both pathways activating MAPK-dependent HIN1 expression in tobacco.
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