PMC

Overall structure of PXY^LRR-TDIF. (A) TDIF binds to the concave surface of PXY^LRR. (B) Electrostatic surface of PXY^LRR around the TDIF-binding groove. White, blue and red indicate neutral, positive and negative surfaces, respectively. TDIF is shown in stick. (C) Structural superposition of PXY^LRR-bound TDIF (purple) with FLS2-bound flg22 (pink) and AtPEPR1-bound AtPep1 (light blue). Hyp4, G6, Hyp7 and P9 of TDIF are shown in stick.

The CLE peptide receptors likely share a conserved ligand recognition mechanism. (A) Superposition of the crystal structure of PXY-TDIF with modeled structures of other known CLE receptors. (B) Superposition of the key TDIF-interacting residues of PXY with their equivalents from the modeled structures. These interacting residues are shown in stick. (C) Frequency plots of the TDIF-interacting residues among the known CLE receptors. V3, Hyp4, G6, N8, and N12 are the conserved residues among the CLE peptides (numbering based on TDIF). Numbers at the bottom indicate the positions of the conserved residues in PXY.

Recognition mechanism of TDIF by PXY. (A) Overview interaction of TDIF with PXY^LRR. The side chains of PXY^LRR are shown in yellow orange. Red dashed lines indicate hydrogen bonds or salt bridges. (B) Interaction of the N-terminal side of TDIF with PXY^LRR. (C) Interaction of the central region of TDIF with PXY^LRR. For (B and C), the side chains of some amino acids from TDIF and PXY^LRR are shown in purple and yellow orange, respectively. Red dashed lines indicate hydrogen bonds or salt bridges. (D) Sequence alignment of PXY^LRR with other CLE receptors around the TDIF binding region. Conserved and similar residues are boxed with red ground and red font, respectively. Conserved motifs are boxed with blue frames.

The last residue of TDIF is recognized by a set of highly conserved amino acids of PXY. (A) Detailed interactions of the conserved TDIF^Asn12 with PXY^LRR. The side chains of some amino acids from TDIF and PXY^LRR are shown in purple and yellow orange, respectively. Red dashed lines indicate hydrogen bonds or salt bridges. (B) Sequence alignment of PXY^LRR with other CLE receptors around the TDIF^Asn12-interacting region. (C) The two consecutive LRRs recognizing the last residue of TDIF are stabilized by a conserved disulfide bond. The sulfur atoms are colored in cyan. (D) Sequence alignment of PXY^LRR with other known CLE receptors around the disulfide bond region. For (B and D), conserved and similar residues are boxed with red ground and red font, respectively. Conserved motifs are boxed with blue frames.

TDIF and CLE42 interact with PXY^LRR, PXL1^LRR and PXL2^LRR in vitro. (A) Both GST-TDIF and GST-CLE42 can pull-down the PXY^LRR, PXL1^LRR and PXL2^LRR proteins. The purified PXY^LRR, PXL1^LRR or PXL2^LRR was incubated with GS4B resin bound by GST-TDIF or GST-CLE42. After extensive washing, the GS4B resin-bound proteins were analyzed by SDS-PAGE and detected by Coomassie blue staining. (B) Measurement of binding affinity between PXY^LRR and TDIF by ITC. Top panel: twenty injections of TDIF solution were titrated into PXY^LRR solution in the ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Bottom panel: the binding isotherm for PXY^LRR and TDIF interaction, the integrated heat is plotted against the molar ratio between TDIF and PXY^LRR. Data fitting revealed a binding affinity of about 33 nM. (C) Sequence alignment of CLE peptide family from Arabidopsis thaliana. Conserved and similar residues are boxed with red ground and red font, respectively.

Mutagenesis analysis of PXY-TDIF complex. (A) Effects of TDIF mutations on interaction with PXY^LRR. GST-TDIF and its mutants were individually incubated with the purified PXY^LRR protein and then flowed through GS4B resin bound by GST-TDIF or its mutants. After extensive washing, the GS4B resin-bound proteins were visualized by Coomassie blue staining following SDS-PAGE. 13R had an additional arginine added at the C terminus of TDIF. (B) Effects of PXY mutations on interaction with GST-TDIF. The assay was performed as described in . For (A and B), blue and green arrows indicate PXY^LRR and GST-TDIF, respectively. (C and D) The vascular phenotype of WT and pxy-5 mutant. (E) The PXY genomic sequence completely rescued the phenotype of pxy-5. (F-K) Complementation results of the mutated PXY genes. (C-F) Upper lane: resin-embedded transverse sections of stem vascular bundles of indicated plants. Scale bars: 10 μm. Lower lane: corresponding hand-cut transverse sections of stem vascular bundles. Scale bars: 20 μm. Ph: phloem; C: cambium; Xy: xylem. Red brackets enclose layer structures of cambium, arrows indicate discontinuous cambial cells.