In plants, the plasma membrane (PM)-localized receptors play central roles in perceiving apoplastic ligands and transducing extracellular signals into intracellular responses (Escocard de Azevedo Manhaes et al., 2021). The PM-localized receptors mainly consist of two types, receptor-like kinase (RLK) and receptor-like protein (RLP) (Shiu and Bleecker, 2003; Escocard de Azevedo Manhaes et al., 2021). In the model plant Arabidopsis, around 420 RLKs have been identified (Gou and Li, 2020). A typical RLK contains an ectodomain, perceiving the external signaling molecules, a transmembrane helix, anchoring the protein at the PM, and a cytosolic kinase domain responsible for activating cellular signaling cascades by phosphorylating downstream substrates. Numerous RLKs have been well functionally characterized to be extensively engaged in almost all aspects during the entire life span of plants, including hormonal pathways, pathogen perception, peptide signalings, and biotic and abiotic responses (Clark et al., 1996, 1997; Li and Chory, 1997; Gomez-Gomez and Boller, 2000; Li et al., 2002; Nam and Li, 2002; Soltabayeva et al., 2022).

Different from RLKs, RLPs only possess an extracellular domain and a transmembrane domain without an enzymatic domain in cytosol (Wang et al., 2008). Instead, RLPs often contain a short carboxyl-tail (C-tail) in cytoplasm. RLPs thus appear to be capable of recognizing ligands but fail to trigger cellular signaling unless they associate with RLKs and function in RLK-containing receptor complexes (Gust and Felix, 2014). The majority of RLPs identified in Arabidopsis contain leucine-rich repeats (LRRs) in the extracellular domain. To date, over 50 LRR-RLP genes have been identified in Arabidopsis genome (Wang et al., 2008; Jamieson et al., 2018). RLPs are far less characterized compared to RLKs in plants. Most RLPs studied so far are found to play important roles in pattern-triggered immunity (PTI) response by perceiving pathogen-derived elicitors (Escocard de Azevedo Manhaes et al., 2021). Only two RLPs have been well studied in regulating plant growth and development. CLAVATA2 (CLV2), also known as RLP10, acts as a critical receptor in modulating the shoot apical meristem (SAM) (Kayes and Clark, 1998). By interacting with a receptor-like cytoplasmic kinase (RLCK), CORYNE (CRN), CLV2 perceives peptide hormone CLV3 to regulate SAM maintenance (Bleckmann et al., 2010). Mutations in CLV2 cause enlarged SAM in plants (Kayes and Clark, 1998). TOO MANY MOUTHS (TMM), also termed RLP17, associates with RLKs ERECTA (ER)/ER-LIKE (ERL) to recognize protein ligand EPIDERMAL PATTERNING FACTOR1/2 (EPF1/2) during guard cell formation (Yang and Sack, 1995; Nadeau and Sack, 2002; Shpak et al., 2005; Lin et al., 2017). Loss-of-function mutants of TMM exhibit significantly increased numbers of stomata in the epidermal cells (Nadeau and Sack, 2002). Of note, few additional RLPs have been found to regulate plant growth and development in the past two decades. Recent studies indicated RLP44 contributes to BR signaling and xylem formation by connecting Brassinosteroid Insensitive1 (BRI1) and Phytosulfokine Receptor (PSKR) (Wolf et al., 2014; Holzwart et al., 2018, 2020; Garnelo Gomez et al., 2021).

Brassinosteroids (BRs) are a group of major phytohormones that play an essential role in regulating plant growth and development, photomorphogenesis, skotomorphogenesis, senescence, productive ability, abiotic stress, and innate immunity (Nolan et al., 2020). Failing to synthesize sufficient BRs, disruption in perceiving BR molecules, or block of BR signaling leads to severe defective phenotypes in plants, featured by dwarfed inflorescence, compact rosette leaves, reduced root growth, and prolonged life span (Nolan et al., 2020). Different from the steroid hormones in animals that are often recognized within the cell, extracellular BRs are perceived by ligand-binding receptor BRI1 and co-receptor BAK1, two LRR-RLKs that are localized at PM (Li et al., 2002; Nam and Li, 2002). The binding of BRs to the extracellular domain of BRI1 creates an interacting surface that recruits the ectodomain of BAK1 to form receptor–BR–coreceptor complex (Santiago et al., 2013; Sun et al., 2013). BRs thus function as molecular glue to induce receptor dimerization. BRI1–BAK1 receptor dimmer formation, therefore, serves as an indispensable event for BR recognition and signaling initiation. BRI1–BAK1 complex is finely tuned by multiple regulators. For instance, BRI1 Kinase Inhibitor1 (BKI1) was identified as a BRI1-interacting protein and functions as a BRI1 repressor (Wang and Chory, 2006). BKI1 is phosphorylated upon BR perception and is disassociated from BRI1 to release its inhibition on BRI1 (Wang et al., 2014). An RLK, BAK1-Interacting Receptor-like Kinase3 (BIR3), and two E3 ligases, Plant U-box E3 ubiquitin ligases12/13 (PUB12/13), were also found to negatively regulate BR signaling by affecting BR receptors (Imkampe et al., 2017; Zhou et al., 2018). A recent study revealed that copine proteins, BONZAI (BON) proteins, facilitate BRI1–BAK1 interaction and transphosphorylation in the presence of Brassinolide (BL), the most active form of BR, to positively regulate BR pathway (Jing et al., 2024).

BAK1 belongs to an RLK family named Somatic Embryogenesis Receptor Kinase (SERK) and is also known as SERK3 (Schmidt et al., 1997). Besides its role in BR signaling, BAK1 and its homologs function as essential components in plant immunity. Acting as a co-receptor, BAK1 associates with multiple pattern recognition receptors (PRRs) to detect a variety of PAMPs in PTI initiation (Chinchilla et al., 2007; Heese et al., 2007; Couto and Zipfel, 2016; Ma et al., 2016). In addition, our previous studies indicated that BAK1 is crucial for effector-triggered immunity (ETI) response. The simultaneous mutations in both BAK1 and BAK1-like (BKK1), the closest homolog of BAK1 and also known as SERK4, lead to spontaneous cell death and lethal phenotypes (He et al., 2007). Our recent work revealed that the autoimmune response in bak1 bkk1 double mutant is caused by constitutively active nucleotide-binding leucine-rich repeat (NLR) proteins, the receptors of ETI responses. BAK1 thus plays a central role in linking PTI and ETI in plant innate immunity (Wu et al., 2020).

In this study, in order to identify potential RLPs involved in BAK1-mediated signaling pathways, we overexpressed all LRR-RLP genes in bak1-3 bkk1-1 background to screen genetic enhancers and suppressors. bak1-3 bkk1-1 is a weak double mutant that shows an obvious cell death phenotype but is completely fertile (He et al., 2007; Wu et al., 2020). Both BR signaling and immune response are disrupted in bak1-3 bkk1-1. We found overexpression of RLP51 was able to obviously suppress BR mutant phenotype, while the cell death symptom was less affected in bak1-3 bkk1-1. The loss-of-function mutations in RLP51 caused enhanced BR mutant phenotypes in bri1-301 and bak1-4 serk1-8. Biochemical assays indicated RLP51 interacted with both BRI1 and BAK1 in vivo. Furthermore, we found the presence of RLP51 strikingly promoted the protein abundances of both BRI1 and BAK1. Interestingly, RLP51 seemed to affect the syntheses, but not the stabilities, of BRI1 and BAK1. Our study thus indicated RLP51 acts as a positive regulator in BR signaling pathway via associating with BRI1 and BAK1 and promoting their abundances.