The cell surface leucine-rich repeat receptor for AtPep1, an endogenous peptide elicitor in Arabidopsis, is functional in transgenic tobacco cells

Abstract

AtPep1 is a 23-aa endogenous peptide elicitor from Arabidopsis leaves that signals the activation of components of the innate immune response against pathogens. Here, we report the isolation of an AtPep1 receptor from the surface of Arabidopsis suspension-cultured cells. An (125)I-labeled AtPep1 analog interacted with suspension-cultured Arabidopsis with a K(d) of 0.25 nM, and an (125)I-labeled azido-Cys-AtPep1 photoaffinity analog specifically labeled a membrane-associated protein of approximately 170 kDa. The labeled protein was purified to homogeneity, and its tryptic peptides were identified as gene At1g73080, which encodes a leucine-rich repeat receptor kinase, here called PEPR1. Verification of the binding protein as the receptor for AtPep1 was established by demonstrating the loss of function of microsomal membranes of two SALK insertional mutants and by a gain in function of the alkalinization response to AtPep1 by tobacco suspension-cultured cells expressing the At1g73080 transgene. Synthetic homologs of AtPep1, deduced from the C termini of six known paralogs of PROPEP1, were biologically active and were competitors of the interaction of an AtPep1 radiolabeled analog with the receptor. The data are consistent with a role for PEPR1 as the receptor for AtPep1 to amplify innate immunity in response to pathogen attacks.

Fig. 1.

Kinetic analysis of ¹²⁵I₁-Tyr-AtPep1 binding to Arabidopsis cells. (A) Tyr-AtPep1 was synthesized and iodinated to produce mono- and diiodinated TyrAtPep1 (I₁-Tyr-AtPep1 and I₂-Tyr-AtPep1, respectively). (B) Biological activity of AtPep1 and analogs from A were assayed in the medium alkalinization assay by using Arabidopsis suspension-cultured cells. (C) Time course of binding of ¹²⁵I₁-Tyr-AtPep1 to Arabidopsis cells. Specific binding was calculated by subtracting nonspecific binding (binding in the presence of 250-fold nonradioactive AtPep1) from total binding in the absence of AtPep1. Error bars indicate standard deviation. (D) Saturation analysis of ¹²⁵I₁-Tyr-AtPep1 binding to Arabidopsis cells. A representative plot from four repetitions is shown. (E) Scatchard analysis of the data from D.

Fig. 2.

Photoaffinity labeling of an AtPep1-binding protein on the surface of Arabidopsis suspension-cultured cells. (A) Construction of the photoaffinity analog azido-Cys-AtPep1. AtPep1 was lengthened at its N terminus with a Cys residue, and the analog was azido-labeled under red light by using the photoaffinity cross-linker N-(4-[p-azidosalicylamido]butyl)-3′-(2′-pyridyldithio)propionamide through the formation of a disulfide bond. (B) Concentration dependence of the alkalinization activity of azido-Cys-AtPep1. (C) Specific binding of 0.25 nM ¹²⁵I-azido-Cys-AtPep1 to Arabidopsis cells under red light in the presence of increasing concentrations of unlabeled AtPep1 (Left) or tomato systemin (Right). Cells were incubated with ¹²⁵I-azido-Cys-AtPep1 for 10 min, washed with water, and irradiated with UV-B for 10 min, and the proteins were separated by SDS/PAGE. The labeled proteins in the gels were identified by exposure to x-ray film. (D) Suramin inhibits ¹²⁵I-azido-Cys-AtPep1-labeling of the 170-kDa binding protein of Arabidopsis suspension-cultured cells. (E) Removal of N-linked glycans with the endoglycosidase peptide-N-glycosidase F (PNGaseF) caused a shift in mobility on SDS/PAGE of ≈12% of the mass of the 170-kDa labeled protein.

Fig. 3.

Purification and identification of an AtPep1-binding protein. (A) (Left) SDS/PAGE analysis of photoaffinity-labeled proteins during purification (described in Materials and Methods). Protein staining is as follows: lane 1, crude extract; lane 2, microsomal fraction; lane 3, the eluate from preparative SDS/PAGE; lane 4, eluate from Con A Sepharose. (Right) Radioautography of gels from Left. (B) Mass spectral analysis of peptides resulting from tryptic digest of the purified, radiolabeled AtPep1-binding protein from lane 4 in A (probability-based molecular weight search score = 609; P = <0.05). Peptide fragments marked with an asterisk matched predicted amino acid sequences of Arabidopsis gene At1g73080. (C) The phylogenetic relationships are based on comparison of the amino acid sequence of PEPR1 with the gene products of other members of the LRR XI subfamily of Arabidopsis LRR receptor-like protein kinases.

Fig. 4.

Confirmation of the functionality of At1g73080 as coding for the AtPep1 receptor PEPR1. (A) (Upper) The location of the T-DNA in each insertional line, SALK_059281 and SALK_014538. (Lower) RT-PCR expression analyses of At1g73080 mRNA in leaves of the following: lane 1, wild-type plants; lane 2, SALK_014538 line; lane 3, SALK_059281 line; lane 4, SALK_064539 line, which is a control line unrelated to At1g73080 in which the glycerophosphoryl diester phosphodiesterase (At5g55480) gene is disrupted by T-DNA. Expression of the β-tubulin gene was used as a control. (B) Photoaffinity labeling under red light of microsomal fractions prepared from 5-week-old plants. Microsomal proteins were photoaffinity-labeled in the presence or absence of 250-fold unlabeled AtPep1 as a competitor. Lane 1, wild-type plants; lane 2, SALK_014538 line; lane 3, SALK_059281 line; lane 4, SALK_064539 line. (C) (Upper) Composition of the vector construct that was used to transform tobacco suspension-cultured cells to express At1g73080. (Lower) RT-PCR analyses of At1g73080 mRNA in wild-type cells and in three transgenic cell lines (#2, #3, and #4). Expression of the elongation factor 1α (EF-1α) gene was used as a control. (D) Alkalinization responses of wild-type and transgenic tobacco suspension-cultured cells to increasing concentrations of AtPep1. Only the transgenic cells responded to AtPep1 by alkalinating the culture medium.

Fig. 5.

Comparisons of the alkalinization activities of 23-aa peptides synthesized from the C-terminal sequences of the proteins encoded by the seven PROPEP paralogs with their abilities to compete with ¹²⁵I₁-Tyr-AtPep1 for binding on the cell surface of suspension-cultured Arabidopsis cells. (A) Medium alkalinization of suspension-cultured Arabidopsis cells by the synthetic AtPep peptides deduced from each of the seven paralogs, assayed at the four concentrations shown. (B) Assays using each synthetic AtPep analog in competition with ¹²⁵I₁-Tyr-AtPep1 for binding to Arabidopsis suspension-cultured cells (for details, see Materials and Methods). Each peptide was incubated with cells at the concentrations indicated just before adding 0.25 nM ¹²⁵I₁-Tyr-AtPep1. After 2 min, the cells were recovered and washed, and the bound radioactivity was quantified.