Signature motif-guided identification of receptors for peptide hormones essential for root meristem growth

Abstract

Peptide-mediated cell-to-cell signaling has crucial roles in coordination and definition of cellular functions in plants. Peptide-receptor matching is important for understanding the mechanisms underlying peptide-mediated signaling. Here we report the structure-guided identification of root meristem growth factor (RGF) receptors important for plant development. An assay based on a signature ligand recognition motif (Arg-x-Arg) conserved in a subfamily of leucine-rich repeat receptor kinases (LRR-RKs) identified the functionally uncharacterized LRR-RK At4g26540 as a receptor of RGF1 (RGFR1). We further solved the crystal structure of RGF1 in complex with the LRR domain of RGFR1 at a resolution of 2.6 Å, which reveals that the Arg-x-Gly-Gly (RxGG) motif is responsible for specific recognition of the sulfate group of RGF1 by RGFR1. Based on the RxGG motif, we identified additional four RGFRs. Participation of the five RGFRs in RGF-induced signaling is supported by biochemical and genetic data. We also offer evidence showing that SERKs function as co-receptors for RGFs. Taken together, our study identifies RGF receptors and co-receptors that can link RGF signals with their downstream components and provides a proof of principle for structure-based matching of LRR-RKs with their peptide ligands.

Figure 1

RxR motif-based identification of the LRR-RK At4g26540 as a receptor of RGF1 in vitro. (A) The RxR motif is used by the LRR-RK AtPEPR1 to recognize AtPep1. Left, overall structure of AtPep1-PEPR1^LRR complex. Right, detailed interactions of the boxed region in the left panel. The side chains of the last residue from AtPep1 and the RxR motif from PEPR1 are labeled. Color codes are indicated. (B) Sequence alignment of LRRs harboring the RxR motif among the LRR XI subfamily of LRR-RKs in Arabidopsis. (C) A schematic diagram showing RxR motif-based identification of the peptide-receptor pair. A purified extracellular LRR domain of an XI LRR-RK is incubated with a pool of peptides (box I) and then subjected to gel filtration (box II). The peak fraction is collected for MS (box III). The peptide identified by MS is regarded as the potential ligand for the LRR-RK (box IV). (D) RGF1 interacts with the LRR domain of the LRR-RK At4g26540 (RGFR1) in vitro. Quantification of the binding affinity between RGF1 and RGFR1^LRR by MST. Data points indicate the differences in normalized fluorescence (%) generated by RGF1 binding to RGFR1^LRR, and curves indicate the calculated fits. Error bars represent standard error of 3 independent measurements. dRGF1: non-sulfated RGF1. (E) RGFR1 and AtPEPR1 share a conserved ligand recognition mode. Left, structural superposition of the RGF1-RGFR1^LRR and AtPep1-AtPEPR1^LRR complexes. Right, detailed interactions of the last residues of RGF1 and AtPep1 with their respective receptors. The side chains of the last residue of RGF1 and the RxR motif of RGFR1 are labeled.

Figure 2

Recognition mechanism of RGF1 by RGFR1. (A) Left, overall structure of the RGF1-RGFR1^LRR complex shown in cartoon. RGFR1^LRR and RGF1 are shown in lemon and pink, respectively. Right, finally refined electron density “2Fo-Fc” contoured at 1.20 σ surrounding RGF1. HyP: hydroxylated proline; sY: sulfated tyrosine. (B) RGF1 binds to a charged surface groove at the inner side of the RGFR1^LRR solenoid. RGFR^LRR is shown in electrostatic surface and RGF1 is shown in cartoon. Red, blue and white indicate negative, positive and neutral surfaces, respectively. (C) Recognition of the last residue of RGF1 by RGFR1^LRR. Red dashed lines indicate hydrogen bonds or salt bridges. (D) Recognition of the sulfated RGF1^Tyr2 by RGFR1. The side chains of RGFR1^LRR and RGF1 are shown in green and pink, respectively. (E) Recognition of the central part of RGF1 by RGFR1^LRR. The side chains of RGFR1^LRR and RGF1 are shown in cyan and pink, respectively. (F) Sequence alignment of the LRRs (the sixth LRR) containing the RxGG motif among the XI subfamily of LRR-RKs in Arabidopsis. The RxGG motif is highlighted within the red square. Five genes containing the RxGG motif were identified as receptors of RGFs and named RGFRs 1-5, and the subfamily of these five genes was named RGFR subfamily.

Figure 3

Expression patterns of RGFRs in root. The expression patterns of RGFR1p::GUS, RGFR2p::GUS, RGFR3p::GUS, RGFR4p::GUS and RGFR5p::GUS, in green seedlings (top) and root tips (bottom).

Figure 4

Loss-of-function rgfr mutants exhibit defects in root meristem and deceased sensitivity to RGF1. (A) Confocal images of the root meristem of Col-0 and mutants (5 DAG stage). The white arrowhead indicates the boundary of meristematic zone and elongation zone. Scale bar, 50 μm. (B) Root lengths of Col-0 and mutant seedlings (7 DAG stage). (C) Changes of root lengths of Col-0 and rgfr mutants in response to RGF1. Quantification of root lengths of Col-0 and mutant seedlings (7 DAG stage) grown in the medium supplied with 1 nM RGF1. (D) Quantification of root meristem cells of Col-0 and mutant seedlings (5 DAG stage) grown in the medium supplied with 1 nM RGF1 or dRGF1. n = 25. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 by Student's t-test. NS indicates not significant.

Figure 5

Identification of SERKs as co-receptors for RGF1. (A) RGF1 induces RGFR1^LRR-SERK1^LRR interaction in vitro. Left, superposition of the gel filtration chromatograms of the RGFR1^LRR+SERK1^LRR and RGFR1^LRR+RGF1+SERK1^LRR proteins. The vertical and horizontal axes represent UV absorbance (280 nm) and elution volume (ml), respectively. Right, coomassie blue staining of the peak fractions shown on the left following SDS-PAGE. M, molecular weight ladder (kDa). (B) RGF1 induces RGFR2^LRR-SERK1^LRR interaction in vitro. Left, superposition of the gel filtration chromatograms of the RGFR2^LRR+SERK1^LRR and RGFR2^LRR+RGF1+SERK1^LRR proteins. (C) RGF1 promotes RGFR1^ΔKD-SERK1^ΔKD/SERK2^ΔKD/BAK1^ΔKD interaction in Nicotiana Benthamiana. Agrobacteria harboring the indicated constructs were syringe infiltrated into tobacco leaves. Peptides (10 nM RGF1, 1 μM PSK as a negative control) were added 2 h before tobacco tissues were harvested for immunoprecipitation with anti-GFP antibody. Immunoblot assays were performed to determine the levels of expressed proteins. Each assay was repeated three times. (D) RGF1 promotes RGFR3^ΔKD-SERK1^ΔKD, RGFR4^ΔKD-SERK1^KD and RGFR5^ΔKD-SERK1^ΔKD interaction in Nicotiana Benthamiana. (E) Confocal images of the root meristem of Col-0 and mutants (5 DAG stage). The white arrowheads indicate the boundary of meristematic zone and elongation zone. Scale bar, 50 μm. (F) Changes in meristematic cortex cell numbers in Col-0 and serk mutants in response to RGF1. Quantification of meristematic cortex cell number of Col-0 and mutant seedlings (5 DAG stage) grown in the medium supplied with 1 nM RGF1. n = 25. (G) Root lengths of Col-0 and serk mutant seedlings (7 DAG stage). (H) Changes in root lengths of Col-0 and serk mutants in response to RGF1. Quantification of root lengths of Col-0 and mutant seedlings (7 DAG stage) grown in the medium supplied with 1 nM RGF1. n = 25. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 by Student's t-test. NS indicates not significant.

Figure 6

Model of RGF signaling initiation. Cartoon illustration showing RGF-induced RGFR activation. The N- (blue) and C-terminal (red) sides of an RGF interact with the RGF-specific motif RxGG and the peptide-determining motif RxR of an RGFR, respectively. The surface formed by the conserved RxR motif of the RGFR and the last residue of the RGF could recruit a SERK member as a co-receptor.