The Medicago truncatula LYR4 intracellular domain serves as a scaffold in immunity signaling independent of its phosphorylation activity

Abstract

Plants engage in a wealth of interactions with beneficial and pathogenic bacteria and fungi and therefore need to monitor their surroundings. To this end, cell-surface receptors, such as lysin motif (LysM) receptors, perceive microbe-associated molecular patterns (MAMPs) and elicit immunity responses or initiate symbiotic associations (Buendia et al., 2018). LysM receptors have an ectodomain built of three interconnected LysM domains and most feature a single transmembrane helix and either an active kinase or a pseudokinase intracellular domain (Gust et al., 2012). The main ligands of LysM receptors are carbohydrates containing N-acetylglucosamine moieties, namely chitooligosaccharides (CO; chitin), lipochitooligosaccharides (LCO), and peptidoglycan (Willmann et al., 2011; Gust et al., 2012; Bozsoki et al., 2017, 2020; Buendia et al., 2018; Gysel et al., 2021).

In the model legume Lotus japonicus (Lotus), the LysM receptor CERK6 (previously called LYS6) and the tandem duplicated receptors LYS13 and LYS14 are involved in chitin-triggered immunity (Bozsoki et al., 2017). LYS13 and LYS14 are classified as pseudokinases, and their expression increases in roots and shoots upon chitin treatment of the plant (Lohmann et al., 2010; Ruman & Kawaharada, 2023). Single mutants of lys13 and lys14 respond to chitin similarly to the wild-type, suggesting functional redundancy between LYS13 and LYS14, whereas cerk6 cannot produce reactive oxygen species (ROS) upon chitin treatment and upregulation of defense-response genes and phosphorylation of MAPK3/6 (mitogen-activated protein kinases) are impaired (Bozsoki et al., 2017). Further investigation of chitin-triggered immunity signaling in Lotus is difficult, as a lys13 lys14 double mutant is not available.

In the model legume Medicago truncatula (Medicago), CERK1 (previously called LYK9) is a homolog of Lotus CERK6 and LYR4 is a homolog of Lotus LYS13 and Lotus LYS14 (Bozsoki et al., 2017; Buendia et al., 2018; Feng et al., 2019). Medicago CERK1 (CERK1 hereafter) and Medicago LYR4 (LYR4 hereafter) have been reported to be indispensable for chitin-triggered immunity with both cerk1 and lyr4 mutants having an increased lesion size upon infection of leaves with Botrytis cinerea (Bozsoki et al., 2017). In addition, ROS responses were absent and phosphorylation of MAPK3 and MAPK6 decreased upon chitin octamer (CO8) treatment in both mutants compared to the wild-type (Bozsoki et al., 2017; Zhang et al., 2024).

CERK1 has an active kinase intracellular domain containing all canonical motifs of a eukaryotic protein kinase, while LYR4 is a classified pseudokinase due to the degeneration of several key kinase motifs: a truncated glycine-rich loop, the DFG-motif NFG, and the HRD-motif HKN (Buendia et al., 2018; Ruman & Kawaharada, 2023; Fig. 1a). Canonically, the glycine-rich loop and the regulatory lysine on the β3-strand stabilize phosphates of bound ATP. Aspartate from the DFG-motif is important for positioning of phosphates for phosphate transfer and the arginine and the aspartate from the HRD-motif contribute to ordering of the activation loop and activation of the incoming substrate, respectively (Taylor & Kornev, 2011; Taylor et al., 2021).

In this study, we investigate how the pseudokinase domain of the Medicago LYR4 LysM receptor kinase mediates downstream signaling in immunity. We determine the crystal structure of the LYR4 intracellular domain with an ATP-analog bound in a noncanonical manner and show that it is catalytically active despite its lack of canonical kinase features. However, in planta experiments demonstrate that the phosphorylation ability is not necessary for the function of LYR4 in chitin-triggered ROS production, but that the presence of its intracellular domain is indispensable. Thus, in chitin-triggered immunity the LYR4 intracellular domain serves as a signaling scaffold independent of its catalytic activity.

Pseudokinases comprise up to 17% of the plant kinome and are crucial for signaling in numerous pathways, for example, those dependent on LysM receptors (Kwon et al., 2019). However, the understanding of pseudokinase signaling mechanisms in plants remains limited. In this study, we therefore investigate the function of Medicago LYR4 in chitin-triggered immunity.

We show that LYR4 can function in perception of chitin in the absence of CERK1, although resulting ROS levels are attenuated. Previously, both the cerk1 and the lyr4 mutants were shown to completely lack a ROS response upon CO8 treatment (Bozsoki et al., 2017; Zhang et al., 2024). The lower ROS response in cerk1 than in the wild-type could be due to LYR4 pairing with a less efficient or less abundant signaling partner. CERK1 is part of the LYK subfamily of LysM receptor-like kinases harboring potentially active kinases, which has 11 members in Medicago, while LYR4 is part of the LYR subfamily that also has 11 members in Medicago, with LYR7 being the phylogenetically closest (Buendia et al., 2018). Recently, Zhang et al. showed that Medicago LYK8 is to some extent functionally redundant with CERK1 in arbuscular mycorrhizal symbiosis. Some of the LysM receptor kinases, which have not been investigated in detail yet, might also have overlapping capacities with the described receptors and could potentially take over their functions.

To better understand signaling mediated by a pseudokinase, we determined the crystal structure of the LYR4 kinase with a bound ATP-analog. The nucleotide was clearly defined in the electron density, which prompted us to investigate the functional relevance of nucleotide binding and phosphotransfer ability. Thermostability assays additionally confirmed that LYR4 kinase binds ATP and closer inspection of the structure showed that the ATP-analog is bound in a noncanonical manner. Despite lacking canonical binding motifs, several pseudokinases have demonstrated the ability to bind nucleotides through various mechanisms and the binding event itself has been proposed to lead to a conformational regulation (Zeqiraj & Van Aalten, 2010; Mace & Murphy, 2021; Sheetz & Lemmon, 2022). In vitro kinase activity assays showed that LYR4 has both auto- and transphosphorylation activity making it an active pseudokinase. In general, many classified pseudokinases are active when tested experimentally, as they make up for lacking motifs by employing modified catalytic mechanisms (Zeqiraj & Van Aalten, 2010; Dar, 2013).

We investigated whether LYR4 is dependent on conformational changes prompted by nucleotide binding or on kinase activity for signaling in immunity. Complementation studies in hairy roots demonstrated that neither ATP-binding nor kinase activity is crucial for LYR4 signaling in chitin-induced ROS production. It is intriguing that LYR4 kinase has retained nucleotide binding ability despite the apparent lack of functional relevance, and we cannot exclude the possibility that nucleotide binding plays a role in other signaling pathways. However, we show that the presence of the LYR4 intracellular domain is indispensable for full ROS elicitation upon chitin treatment (Fig. 2d). We conclude that LYR4 pseudokinase serves a noncatalytic scaffold function in immunity signaling and that CERK1 kinase activity is responsible for signaling downstream of the putative CERK1–LYR4 receptor complex. In a broader view, scaffolding pseudokinase receptors like LYR4 might be necessary to correctly arrange their co-receptors, as well as mediate interactions with downstream signaling partners. Pseudokinases have been shown to often be as dynamic as typical kinases, which allows them to act as switches or allosteric regulators while acting as scaffolds for complex assembly (Sheetz et al., 2020; Mace & Murphy, 2021; Sheetz & Lemmon, 2022; Mühlenbeck et al., 2023). Future studies will elucidate how kinase and pseudokinase receptor complexes are formed to mediate signaling.

None declared.

BS, HR, MVK, MML, CK and GK were involved in investigation. BS, HR, KG, ML, JS, SR and KRA were involved in formal analysis. SF and FF were involved in resources. BS and HR were involved in visualization. SR and KRA were involved in conceptualization. KG, SR and KRA were involved in supervision. KRA was involved in project administration. GEDO, SR and KRA were involved in funding acquisition. BS, HR and KRA were involved in writing – original draft preparation. All authors were involved in writing – review and editing. BS and HR contributed equally to this work.

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