Subfamily C7 Raf-like kinases MRK1, RAF26, and RAF39 regulate immune homeostasis and stomatal opening in Arabidopsis thaliana

Introduction

Plants encounter a variety of stressors in the environment that can negatively impact their growth and survival. The ability of plants to respond to danger signals such as drought, heat, cold, salinity, or pathogen attack, is critical to optimizing growth and reproduction in a changing environment. Lacking a humoral system, plants rely on innate and cell-autonomous immune responses to fight against disease. Plant cell membranes contain high-affinity transmembrane pattern recognition receptors (PRRs) that detect highly conserved microbial molecules known as microbe-associated molecular patterns (MAMPs) or endogenous damage-associated molecular patterns (DAMPs). Small peptides known as phytocytokines can also be secreted into the extracellular space, bind PRRs, and potentiate immune signaling (Gust et al., 2017; Segonzac & Monaghan, 2019). Plant PRRs are typically receptor kinases (RKs) or receptor-like proteins (RPs). RKs contain a ligand-binding ectodomain, a transmembrane domain, and an intracellular protein kinase domain, allowing them to both detect M/DAMPs and transduce the signal through substrate phosphorylation. In contrast to RKs, RPs lack a kinase domain, relying on regulatory RKs to relay the signal (DeFalco & Zipfel, 2021). The largest group of plant PRRs are the leucine-rich repeat (LRR)-containing RKs, which preferentially bind protein-based M/DAMPs. The LRR-RK FLAGELLIN SENSING 2 (FLS2) binds flg22, a 22-amino acid epitope from the N-terminus of bacterial flagellin, while the LRR-RKs EF-Tu RECEPTOR (EFR) and PEP-RECEPTOR 1 and 2 (PEPR1/2) bind the 18-amino acid epitope of elongation factor Tu (elf18) or endogenous peptide AtPep1, respectively (Zipfel et al., 2006; Chinchilla et al., 2007; Krol et al., 2010; Yamaguchi et al., 2010). Both RKs and RPs form heteromeric complexes with regulatory co-receptors at the plasma membrane that typically engage in reciprocal trans-phosphorylation, ultimately leading to receptor complex activation and intracellular signaling, including changes in ion flux, defense gene expression, and ROS production (Couto & Zipfel, 2016).

Many PRRs associate closely with several classes of intracellular protein kinases including receptor-like cytoplasmic kinases (RLCKs) (Liang & Zhou, 2018), mitogen-activated protein kinases (MAPKs) (Taj et al., 2010), and calcium-dependent protein kinases (CDPKs) (Yip Delormel & Boudsocq, 2019). Here we focus on CPK28, a multi-functional CDPK with roles in plant growth and development (Matschi et al., 2013), stress responses (Jin et al., 2017; Hu et al., 2021; S. Ding et al., 2022; Y. Ding et al., 2022), and defense against pathogens (Monaghan et al., 2014, 2015; Matschi et al., 2015). In immune signaling, CPK28 buffers the accumulation of the RLCK BOTRYTIS INDUCED KINASE 1 (BIK1), a common substrate of multiple receptors and a critical signaling node in plant immunity (Monaghan et al., 2014; J. Wang et al., 2018; DeFalco & Zipfel, 2021). CPK28 phosphorylates the E3 ubiquitin ligases PLANT U-BOX 25 (PUB25) and PUB26, enhancing their ability to polyubiquitinate BIK1 resulting in its proteasomal turnover (J. Wang et al., 2018). The CPK28-PUB25/26 regulatory module thus buffers BIK1 protein accumulation to optimize immune output (Dias et al., 2022).

In the current study, we aimed to identify additional CPK28 binding partners in Arabidopsis using a co-immunoprecipitation-based proteomics approach. We found that many protein kinases, including MIXED LINEAGE KINASE/RAF-RELATED KINASE 1 (MRK1) copurify with CPK28-YFP. Metazoan rapidly accelerated fibrosarcoma (Raf) kinases function in MAPK cascades. In mammals, the Ras–Raf–MEK–ERK pathway has been intensely studied and serves as a paradigm for membrane-to-nucleus signal transduction. In this pathway, binding of epidermal growth factor (EGF) to the EGF receptor at the plasma membrane results in activation and phosphorylation of its cytoplasmic kinase domain. This activates the GTPase Ras, which then binds to and activates Raf, which serves as a MAPK kinase kinase (MKKK), phosphorylating and activating a MAPK kinase (MKK), which then phosphorylates and activates a MAPK (originally named extracellular signal regulated kinase; ERK) (Terrell & Morrison, 2019). Reflecting the expansion of the protein kinase family in the plant kingdom, there are 20 MAPKs, 10 MKKs, and 80 MKKKs in Arabidopsis (González-Coronel et al., 2021) – many more than in mammals. Despite their number, very little is known about MKKKs. Sequence homology defines three distinct subclasses known as MKKK, ZIK, and Raf-like kinases. There are 48 Raf-like kinases in Arabidopsis, divided into 11 subfamilies: B1–B4 and C1–C7 (Jonak et al., 2002; González-Coronel et al., 2021). Phylogenetic analyses indicate that plant Raf-like kinases do not cluster with metazoan MKKK or Raf kinases (Tang & Innes, 2002; Champion et al., 2004) and are considered a plant (Pl)-specific family of tyrosine kinase-like (TKL) proteins (TKL-Pl-4) (Lehti-Shiu & Shiu, 2012). Despite this divergence, TKL-Pl-4 kinases share sequence features with metazoan Rafs and MLKs and may therefore function biochemically as MKKKs in MAPK cascades (Champion et al., 2004; Lehti-Shiu & Shiu, 2012; González-Coronel et al., 2021), however, this has not been comprehensively studied.

MRK1 belongs to the C7 subfamily of Raf-like kinases, together with RAF26, RAF39, CONVERGENCE OF BLUE LIGHT AND CO₂ 1 (CBC1), and CBC2 (Hiyama et al., 2017). CBC1 and CBC2 are highly expressed in guard cells and have established roles in light-induced stomatal opening (Hiyama et al., 2017). While stomatal pores play a critical role in controlling gas exchange and water transpiration, they also represent a point of entry for microbial pathogens (Melotto et al., 2006), and immune-induced stomatal closure is a well-documented antimicrobial defense response (Melotto et al., 2017). Here, we define redundant roles for MRK1, RAF26, and RAF39 in the inhibition of immune-triggered production of reactive oxygen species (ROS). We also demonstrate that MRK1, RAF26, and RAF39 function in stomatal opening, which correlates with enhanced resistance to a bacterial pathogen. We show that MRK1, RAF26, and RAF39 localize to endomembranes. We confirm that MRK1, RAF26, and RAF39 associate with CPK28 and that CPK28 can trans-phosphorylate RAF26 and RAF39 in vitro. We further show that MRK1, RAF26, and RAF39 are active kinases that can auto-phosphorylate in vitro. However, they are unable to trans-phosphorylate any of the 10 Arabidopsis MKKs in vitro, suggesting that they possess substrate specificities distinct from canonical MKKKs. Overall, our study reveals that C7 Raf-like kinases are CPK28 substrates that function redundantly in immune-triggered ROS production and stomatal opening and provide evidence that they probably do not function as MKKKs.