Constitutive activation of leucine-rich repeat receptor kinase signaling pathways by BAK1-interacting receptor-like kinase 3 chimera

Abstract

194 words) Receptor kinases with extracellular leucine-rich repeat domains (LRR-RKs) form the largest group of membrane signaling proteins in plants. LRR-RKs can sense small molecule, peptide or protein ligands, and may be activated by ligand-induced interaction with a shape complementary SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptor kinase. We have previously shown that SERKs can also form constitutive, ligandindependent complexes with the LRR ectodomains of BAK1-interacting receptor-like kinase 3 (BIR3) receptor pseudokinases, negative regulators of LRR-RK signaling. Here we report that receptor chimaera in which the extracellular LRR domain of BIR3 is fused to the cytoplasmic kinase domains of the SERK-dependent LRR-RKs BRASSINOSTEROID INSENSITIVE1, HAESA and ERECTA form tight complexes with endogenous SERK co-receptors in the absence of ligand stimulus. Expression of these chimaera under the control of the endogenous promoter of the respective LRR-RK leads to strong gain-of-function brassinosteroid, floral abscission and stomatal patterning phenotypes, respectively. Importantly, a BIR3-GSO1/SGN3 chimera can partially complement sgn3 Casparian strip formation phenotypes, suggesting that GSO1/SGN3 receptor activation is also mediated by SERK proteins. Collectively, our protein engineering approach may be used to elucidate the physiological functions of orphan LRR-RKs and to identify their receptor activation mechanism in single transgenic lines. 2 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 MAP kinase signaling pathway (Meng et al., 2015) with diverse roles in plant development involvesthe LRR-RK ERECTA (ER) and its paralogs ERECTA-LIKE1 (ERL1) and ERL2 (Torii et al., 1996;Shpak, 2013). ER, ERL1 and ERL2 together control stomata development and their correct spacing on the leaf surface (Shpak et al., 2005). Cysteine-rich EPIDERMAL PATTERNING FACTOR peptides (EPFs) bind to the ectodomains of ER, ERL1 and ERL2 which form constitutive complexes with the ectodomain of the receptor-like protein (RLP) TOO MANY MOUTH (TMM) Vl and elution at 0.75 ml min -1 was monitored by ultraviolet absorbance at λ = 280 nm. To chimera; # numbers indicate independent lines.(C) Anti-GFP western blot together with the Ponceau-stained membrane as loading control.

Ligand-recognizing motifs in plant LysM receptors are major determinants of specificity

“…Our results based on native expression of chimeric receptors derived from paralogous proteins showed that ectodomains of NFR1 and CERK6 contain major determinants for ligand perception and signaling specificity that can be further modulated by their intracellular regions. Similar modulation of specificity in the final signaling output by the intracellular domains has been observed with Leucine-rich repeat receptor kinases (34).…”

Pan‐brassinosteroid signaling revealed by functional analysis of NILR1 in land plants

“…A recent study reported that the extracellular LRR domain of BIR3 fused to the cytoplasmic kinase domains of the SERK‐dependent BRI1, HAESA and ERECTA formed tight complexes with endogenous SERK coreceptors in the absence of ligand stimulus, resulting in a strong gain‐of‐function phenotype when expressed (Hohmann et al ., 2020). We generated the chimeric constructs BIR3‐EMS1, BIR3‐NILR1 and BIR3‐BRI1 as reported (Fig.…”

“…An engineered‐inducible dimerization (RiD) receptor system can trigger a receptor‐specific RLK signal independently of their cognate ligands or endogenous receptors (Kim et al ., 2021). Another study has indicated constitutive activation of BIR3 fused to the cytoplasmic kinase domains of the SERK‐dependent RLKs in the absence of the ligand (Hohmann et al ., 2020). In the present study, we generated chimeric receptors using the ECDs and ICDs of NILR1, EMS1 and BRI1 as well as the ECDs of AtBRI1 and ICDs of NILR1 of other plants.…”

The brassinosteroid receptor geneBRI1safeguards cell-autonomous brassinosteroid signaling across tissues

“…In parallel, we tested whether BRI1 kinase acHvity is not only necessary but also sufficient to confer bri 3 rescue. To this end we introduced a consHtuHvely acHve chimeric receptor composed of the BAK1-INTERACTING RECEPTOR-LIKE KINASE 3 (BIR3) extracellular domain and the intracellular BRI1 kinase domain (BIR3 EXT BRI1 INT ) (Hohmann et al, 2020) into the bri 3 background. The BIR3 EX BRI1 INT chimera can trigger strong gain-of-funcHon phenotypes that mimic brassinosteroid pathway hyperacHvity (Hohmann et al, 2020).…”