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CLE42 binding induces PXL2 interaction with SERK2

Shulin Mou,Xiaoxiao Zhang,Zhifu Han,Jiawei Wang,Xinqi Gong,J. Chai

Published 2017 in Protein & Cell

ABSTRACT

Plant tissues derived from meristems including SAM (shoot apical meristem) and RAM (root apical meristem) are located at the tips of shoot and root and procambial cell tissues in the vascular system (Simon and Stahl, 2011). Through asymmetric periclinal cell division, a few layers of stem cells in vascular meristems differentiate into apposing xylem and phloem cells, forming the conducting system and playing an important role in long-distance transport of water, nutrients, sugars, and signaling molecules such as hormones in plant (Elo et al., 2013). The leucine rich repeat receptor kinase (LRR-RK) PXY (phloem intercalated with xylem) belongs to XI subfamily of leucine rich repeat receptor-like kinase (LRR-RLK). PXY is a receptor of CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptide CLE41/44 or TDIF (tracheary elements differentiation inhibitory factor). TDIF-PXY signaling functions to promote procambial cell proliferation and suppress tracheary element differentiation, thus playing an important role in wood formation and vascular (Hirakawa et al., 2008; Ito et al., 2006; Fisher and Turner, 2007). The tdr-1/pxy-5 mutant was severely impaired in the proliferation of procambial cells (Hirakawa et al., 2008). WUSCHEL HOMEOBOX RELATED 4 (WOX4) and WOX14 are downstream components of PXY-TDIF signaling and function redundantly in regulating vascular cell division (Etchells et al., 2013; Hirakawa et al., 2010). CLE41/44 has 12 aa (His-Glu-Val-Hyp-Ser-Gly-Hyp-Asn-Pro-Ile-Ser-Asn) in its mature form. A recent structural study revealed that the last amino acid of the peptide is required for CLE41/44 recognition by PXY (Zhang et al., 2016b). PXL1 (PXY-like 1) and PXL2 (PXY-like 2) are two closely related LRR-RKs to PXY, sharing 61% and 62% sequence similarity with PXY, respectively. However, in contrast with PXY, neither pxl1 nor pxl2 plants displayed an obvious phenotype in the vascular stem (Fisher and Turner, 2007). Nonetheless, simultaneous mutations of the three LRR-RKs genes (pxy-3 with pxl1 and pxl2) generated an enhanced vascular phenotype observed in pxy-3 plants with flatter vascular bundles and a less clear distinction between xylem and phloem. These results suggest that PXL1 and PXL2 can function redundantly or synergistically with PXY in regulating vascular-tissue development. Indeed, biochemical data showed that CLE41/44 also interacted with PXL1, though with a lower affinity than that of CLE41/44 with PXY (Zhang et al., 2016b). Interestingly, the triple-mutant did not display a more pronounced phenotype than the pxl1 and pxl2 plants, suggesting that these two genes might also have a different role from PXY in vascular development (Fisher and Turner, 2007). PXL2 belongs to XI LRR-RK subfamily, members of which have been proposed to recognize small signaling peptides through the conserved Arg-x-Arg (RxR, x stands for any amino acid) motif (Zhang et al., 2016a). We therefore reasoned that PXL2 may also recognize a small signaling peptide(s) to mediate vascular development. To test this idea, we purified the extracellular LRR domain protein of PXL2 (PXL2) and incubated the purified protein with a pool of chemically synthesized peptides featuring a free C-terminal histidine or asparagine. The mixture was then subject to gel filtration to separate the PXL2-bound peptide(s) from the others (Fig. 1A). The protocol described previously (Song et al., 2016) was used to detect the peptide (s) bound to the PXL2 protein by mass spectrometry. By using this method, we found that CLE42 was co-purified with the PXL2 protein in the gel filtration assay, suggesting that CLE42 may act as a ligand of PXL2 (Fig. 1B). To further support this conclusion, we assayed the binding affinity of CLE42 with PXL2 using ITC. The ITC results showed that CLE42 bound to the PXL2 protein with a dissociation constant (Kd) of ∼2.75 μmol/L (Fig. 1C). CLE42 is also a dodecapeptide (His-Gly-Val-Hyp-Ser-Gly-Hyp-Asn-Pro-IleSer-Asn) and differs from CLE41 only in the 2nd position. ITC assays indicated that CLE41 also interacted with PXL2 but with a slightly lower affinity (Kd ∼10 μmol/L, Fig. 1D). As a negative control, CLE13 (Arg-Leu-Val-HypSer-Gly-Hyp-Asn-Pro-Leu-His-His) had no detectable interaction with PXL2 as indicated by ITC (Fig. S1). We then solved the crystal structure of PXL2 determined at resolution of 3.6 Å (Fig. 2A). Structural comparison showed that PXL2 and PXY are highly conserved in their structures (Fig. S2A). Although we have not obtained the structure of PXL2 bound by CLE42, the complex can be modeled with high confidence using the structure of PXY-CLE41 as a template given that the conserved

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