Fig. 1: CLE19 interacts directly with PXL1 and induces PXL1 phosphorylation. | Nature Communications

Fig. 1: CLE19 interacts directly with PXL1 and induces PXL1 phosphorylation.

From: PXL1 and SERKs act as receptor–coreceptor complexes for the CLE19 peptide to regulate pollen development

Fig. 1: CLE19 interacts directly with PXL1 and induces PXL1 phosphorylation.The alt text for this image may have been generated using AI.

a Identification of CLE19–PXL1 interaction by gel filtration-MALDI-TOF. b Measurement of binding affinity between CLE19 and PXL1LRR by ITC. Top panel: 20 injections of CLE19 solution were titrated into PXL1LRR solution in the ITC cell. The area of each injection peak corresponds to the total heat released by that injection. Bottom panel: the binding isoform for CLE19 and PXL1LRR interaction. The integrated heat is plotted against the molar ratio of CLE19 to PXL1LRR. Data fitting revealed a binding affinity of ~346 nM. c Dot blot showing the direct interaction between PXL1 and CLE19. The left panel shows increasing concentrations of immobilized 3xFLAG peptide and PXL1-3xFLAG fusion protein on nitrocellulose filter membrane. The right panel shows that PXL1-3xFLAG but not 3xFLAG can bind to CLE19. d–f CLE19 specifically induces the phosphorylation of PXL1 in vivo. d Seedlings of pPXL1::PXL1-FLAG transgenic plants were subjected to CLE19 treatment or left untreated. Electrophoretic mobility of the phosphorylated PXL1-FLAG band (indicated by a red arrowhead) was altered in the phos-tag gel after a 1.5-h incubation with CLE19, which was abolished by treated with λpp. HSP was used to indicate the input amount. The intensity of each band in (d) was measured using ImageJ software. After measurement, the intensity of the non-phosphorylated PXL1-FLAG band in the first lane from left to right was set as a reference value of 1, and the value of each detected band in the second, third and fourth lanes were expressed as the ratio of the detected bands to that of the reference band. Similarly, the intensity of HSP in the first lane from left to right was set to 1, and the intensity of other bands was presented as the ratio to this HSP band. e Seedlings of pPXL1::PXL1-FLAG transgenic plants were treated with 20 μm CLE19 for 1.5 h, and then PXL1-FLAG proteins were pulled down by FLAG beads. The CLE19-induced phosphorylation of PXL1 was verified with a pT/S antibody, and the anti-FLAG antibody was used to indicate the input amount. f Neither CLE19G6T nor CLV3 induced the phosphorylation-related migration of PXL1. In (d–f), the phosphorylated bands are indicated by red arrows, and the unphosphorylated bands are indicated with blue arrowheads. Three times experiments were repeated with similar results for (d–f).

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