Molecular mechanism for the recognition of sequence-divergent CIF peptides by the plant receptor kinases GSO1/SGN3 and GSO2

Satohiro Okuda, Satoshi Fujita, Andrea Moretti, Ulrich Hohmann, Verónica G. Doblas, Yan Ma, Alexandre Pfister, Benjamin Brandt, Niko Geldner, and Michael Hothorn

PNAS February 4, 2020 117 (5) 2693-2703

Significance

Plants have evolved unique membrane receptor kinases with extracellular leucine-rich repeat domains that regulate diverse developmental processes and that form the first layer of the plant immune system. Here it is shown that 2 sequence-related receptor kinases and their shape-complementary coreceptors selectively sense members of a small family of secreted peptide hormones to control formation of an important diffusion barrier in the plant root.

Abstract

Plants use leucine-rich repeat receptor kinases (LRR-RKs) to sense sequence diverse peptide hormones at the cell surface. A 3.0-Å crystal structure of the LRR-RK GSO1/SGN3 regulating Casparian strip formation in the endodermis reveals a large spiral-shaped ectodomain. The domain provides a binding platform for 21 amino acid CIF peptide ligands, which are tyrosine sulfated by the tyrosylprotein sulfotransferase TPST/SGN2. GSO1/SGN3 harbors a binding pocket for sulfotyrosine and makes extended backbone interactions with CIF2. Quantitative biochemical comparisons reveal that GSO1/SGN3–CIF2 represents one of the strongest receptor–ligand pairs known in plants. Multiple missense mutations are required to block CIF2 binding in vitro and GSO1/SGN3 function in vivo. Using structure-guided sequence analysis we uncover previously uncharacterized CIF peptides conserved among higher plants. Quantitative binding assays with known and novel CIFs suggest that the homologous LRR-RKs GSO1/SGN3 and GSO2 have evolved unique peptide binding properties to control different developmental processes. A quantitative biochemical interaction screen, a CIF peptide antagonist and genetic analyses together implicate SERK proteins as essential coreceptor kinases required for GSO1/SGN3 and GSO2 receptor activation. Our work provides a mechanistic framework for the recognition of sequence-divergent peptide hormones in plants.

See: https://www.pnas.org/content/117/5/2693

Figure 7: A quantitative interaction screen identifies SERK proteins as putative coreceptors for GSO1/SGN3. (A) Schematic overview of the biochemical screen for a GSO1/SGN3 coreceptor. Streptavidin (in gray) was immobilized by using amine coupling as described in Fig. 1A. The GSO1/SGN3 ectodomain was captured by streptavidin on the GCI chip surface (in blue), the CIF peptide is provided in access in the running buffer (in black), and different recombinantly purified coreceptor candidates are assayed for binding (in orange). (B) Coomassie-stained SDS/PAGE of the receptor and coreceptor candidates used in the screen. Each lane depicts 1 µg of the LRR ectodomain of each indicated candidate. Shown are isolated monomeric peak fractions from size-exclusion chromatography experiments. (C) GCI assays of SERK1 LRR-RK ectodomain versus the SGN3 wild-type and mutant ectodomains in the presence of CIF2 variant peptides. The remaining candidates are shown in SI Appendix, Fig. S10. Coreceptor candidates were supplied at a flow rate of 25 µL min⁻¹. Sensorgrams are shown with raw data in red and their respective fits in black. A 1-to-1 binding model was used for analysis. Table summaries of kinetic parameters are shown. (D) Complex formation of SERK1 and SGN3 ectodomains. (D, Left) Analytical size-exclusion chromatography traces of the SGN3 ectodomain in the absence (blue line) or presence of CIF2^WT peptides (red dotted line) or CIF2^I81D antagonistic peptides (black dotted line). An SDS/PAGE analysis of the corresponding fractions is shown alongside. The theoretical molecular weight is 94.1 kDa for SGN3 (residues 19 to 870) and 21.5 kDa for SERK1 (residues 24 to 213), respectively. (E) Induced barrier defect in inducible SERK3 dominant negative lines. Quantification of barrier permeability was done using the PI assay (n ≥ 8 for each condition). Shown are box plots spanning the first to third quartiles, with the bold line representing the median and circles indicating the raw data. Whiskers indicate maximum and minimum values, except outliers (b statistically significant difference from a, with P < 0.05, one-way ANOVA and Tukey test).