Tools and Strategies to Match Peptide-Ligand Receptor Pairs

Abstract

Peptide signals have emerged as an important class of regulators in cell-to-cell communication in plants. Several families of small, secreted proteins with a conserved C-terminal Pro-rich motif have been identified as functional peptide signals in Arabidopsis thaliana. These proteins are presumed to be trimmed proteolytically and undergo posttranslational modifications, such as hydroxylation of Pro residues and glycosylation, to form mature, bioactive signals. Identification and matching of such ligands with their respective receptors remains a major challenge since the genes encoding them often show redundancy and low expression restricted to a few cells or particular developmental stages. To overcome these difficulties, we propose the use of ectopic expression of receptor genes in suitable plant cells like Nicotiana benthamiana for testing ligand candidates in receptor output assays and in binding studies. As an example, we used the IDA peptide HAE/HSL2 receptor signaling system known to regulate floral organ abscission. We demonstrate that the oxidative burst response can be employed as readout for receptor activation by synthetic peptides and that a new, highly sensitive, nonradioactive labeling approach can be used to reveal a direct correlation between peptide activity and receptor affinity. We suggest that these approaches will be of broad value for the field of ligand-receptor studies in plants.

Figure 1.

Ox-Burst by the IDA-HAE/HSL2 Signaling Module. (A) Alignment of the C-terminal region of IDA, IDL1-5 from Arabidopsis, and IDA orthologs from other species, CLV3 and CLE2. Sequence similarities within the PIP domain of the IDA peptides are highlighted in green. The invariant proline, hydroxyprolinated in CLV3, CLE2, and presumably also IDA, and the His conserved in all these signal peptides are indicated in black. (B) Protein gel blot of N. benthamiana leaves expressing Halo-tagged HAE and HSL2 as full-length constructs or as kinase truncated versions (ΔKD), respectively. Blots developed with α-Halo antibody stain single bands that migrate with molecular weights that are higher than those anticipated by the polypeptide chains alone. This is likely due to glycosylation of the receptors, as previously reported for HAE (Jinn et al., 2000). (C) IDA triggers the production of ROS through HSL2 in N. benthamiana. Oxidative burst was measured in leaf pieces of untransformed N. benthamiana (control) or leaves transiently expressing the HSL2 receptor or HSL2ΔKD, HAE, or HAEΔKD as indicated. Leaf pieces were exposed to various concentration of the EPIP peptide of IDA (top panel), the PIP peptide (second panel from top), or the peptide PIPPo, PIP with hydroxylation of the conserved Pro at position 7 (bottom two panels). Ox-burst by the luminol-based assay was monitored over time as relative light units (RLU). Error bars indicate sd of n = 3 replicates.

Figure 2.

Acridinium Conjugate of flg22 as a Sensitive Probe to Detect Specific Binding Sites on Tomato Cells. (A) Peptides used in the experiment. Acri-flg22 is flg22 with an acridinium-ester conjugated to its N terminus, and flg22 and flg15 are potent agonistic ligands for the FLS2 receptor, while flg15^Atum has no affinity for this receptor in tomato cells (Meindl. et al., 2000). (B) Binding of Acri-flg22 to intact tomato cells. Aliquots of cells were treated for 5 min with 30 pM Acri-flg22 and a high molar excess of unlabeled flg22, flg15, or flg15^Atum as indicated. After extensive washing, acridinium bound to cells was measured by tracing light emission (relative light units [RLU]) induced by applying 20 mM H₂O₂ in 0.1 M NaOH. (C) Binding competition by different concentration of unlabeled flg22. Cells were treated as in (B) and acri-flg22 bound to cells was quantified by integrating the flash of light emitted after application of 20 mM H₂O₂ in 0.1 M NaOH.

Figure 3.

Binding of the IDA-Derived Peptide Acri-PIPPo to HSL2. (A) Modified IDA peptides bind directly to the ectodomain of HSL2. Leaf material from N. benthamiana expressing HSL2, HAE, or the truncated version HSL2ΔKD or HAEΔKD lacking the kinase domain was incubated with 10 nM acri-PIPPo peptide in the presence or absence of 10 μM unlabeled VPIPPo as a competitor. Controls show binding to leaf material from nontransformed N. benthamiana. Bars and error bars show averages and standard deviations of n = 3 replicates. RLU, relative light units. (B) Saturation of acri-PIPPo binding to the HSL2 receptor. Specific binding of acridinium-tagged VPIPPo was determined by subtracting binding observed with untransformed leaf material at the different concentrations from the values measured for the HSL2 expressing leaf material. (C) Binding kinetics and reversibility of binding to the HSL2 receptor. Plant material from HSL2 expressing or nonexpressing (control) leaf samples were incubated with 10 nM acri-PIPPo. Reversibility of binding was tested by adding 10 μM of VPIPPo (indicated with an arrow) at 25 min. Binding is expressed as percentage of maximal binding in material expressing HSL2. (D) HSL2-Halo expressing leaf material incubated with acri-PIPPo and increasing concentrations of EPIP and PIPPo as competitors. Leaf tissue not expressing the receptor was incubated with the acri-PIPPo peptide and different concentrations of PIPPo as a control to determine background signal. The signal detected from samples without competitor was defined as 100% binding.