生命科学学院戴绍军教授研究团队在Trends in Plant Science期刊上发表论文.pdf

TRPLSC 2444 No. of Pages 19 Trends in Plant Science Feature Review Cysteine-rich receptor-like protein kinases: emerging regulators of plant stress responses Yongxue Zhang, 1,2,6 Haodong Tian, 1,6 Daniel Chen, 3 Heng Zhang, 1 Meihong Sun, 1 Sixue Chen, 4 Zhi Qin, 1,* Zhaojun Ding , 5,* and Shaojun Dai 1,* Cysteine-rich receptor-like kinases (CRKs) belong to a large DUF26-containing receptor-like kinase (RLK) family. They play key roles in immunity, abiotic stress response, and growth and development. How CRKs regulate diverse processes is a long-standing question. Recent studies have advanced our understanding of the molecular mechanisms underlying CRK functions in Ca2+ influx, reactive oxygen species (ROS) production, mitogen-activated protein kinase (MAPK) cascade activation, callose deposition, stomatal immunity, and programmed cell death (PCD). We review the CRK structure–function relationship with a focus on the roles of CRKs in immunity, the abiotic stress response, and the growth–stress tolerance tradeoff. We provide a critical analysis and synthesis of how CRKs control sophisticated regulatory networks that determine diverse plant phenotypic outputs. Highlights Cysteine-rich receptor-like kinases (CRKs) are evolutionarily conserved DUF26-containing receptor-like kinases (RLKs). CRKs regulate pattern-triggered immunity and effector-triggered immunity by modulating reactive oxygen species (ROS) production, Ca2+ influx, mitogenactivated protein kinase (MAPK) cascade activation, phytohormone signaling, and callose deposition. CRKs control abiotic stress response and the stress–growth balance. Future research using CRISPR, inducible systems, single-cell omics, post-translational modification analysis, and proximity-labeling proteomics will advance our understanding of plant CRKs. CRKs are evolutionarily conserved RLKs CRKs belong to a large RLK family containing many evolutionarily conserved members in vascular plants but not in bryophytes and algae [1]. Forty-four CRKs in Arabidopsis thaliana (arabidopsis) and 1074 CRKs from 14 crops have been found through multi-omic and molecular genetic analyses, but only 63 CRKs have been shown to function in regulating plant immunity, abiotic stress (e.g., salinity, osmosis, oxidation, and heat) responses, and growth and development [2–21] (Figure 1, and Table S1 in the supplemental information online). Specifically, CRKs are involved in regulating Ca2+ influx, ROS homeostasis, MAPK cascade activation, and callose deposition, thereby modulating stomatal closure, pathogenesis-related (PR) gene expression, and PCD (Figure 1). Most CRK genes localize in tandem arrays on chromosomes During evolution, CRKs have expanded lineage specifically through relatively recent tandem duplication and preferential retention of duplicates following whole-genome duplication [1]. Some CRKs share a high level of sequence similarity and localize in tandem arrays on chromosome, as found in arabidopsis [22], pepper (Capsicum annuum) [11], cotton (Gossypium barbadense) [13], soybean (Glycine max) [15], and a halophyte pasture alkaligrass (Puccinellia tenuiflora) [21] (Figures 2A and S1A in the supplemental information online). The tandem duplications of CRK evolved mainly through unequal crossover or homologous recombination events [1]. These tandem repeats of CRKs have been suggested to correlate with stress responses to facilitate stressadaptive evolution because most clustered CRKs function in adaptation to environmental stimuli and pathogen infection [1,11,23]. An interesting question concerns whether tandem expansion drives the evolution of CRK functional diversification such as subfunctionalization and neofunctionalization. Answering this question requires a large-scale functional analysis of CRK members. However, only 42 CRKs located in tandem repeats have been characterized (Table S1). How many CRKs are functionally redundant or unique through point mutations, chromosomal deletions, and promoter regulatory elements still needs to be established. Trends in Plant Science, Month 2023, Vol. xx, No. xx 1 Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China 2 Shanghai Key Laboratory of Protected Horticulture Technology, Horticultural Research Institute, Shanghai Academy of Agricultural Science, Shanghai 201403, China 3 MD Program of Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA 4 Department of Biology, The University of Mississippi, Oxford, MS 38677, USA 5 Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, Shandong, China 6 Equal contributions *Correspondence: qinzhi@shnu.edu.cn (Z. Qin), dingzhaojun@sdu.edu.cn (Z. Ding), and daishaojun@shnu.edu.cn (S. Dai). https://doi.org/10.1016/j.tplants.2023.03.028 © 2023 Elsevier Ltd. All rights reserved. 1 Trends in Plant Science Trends in Plant Science Figure 1. Schematic diagram of the regulatory roles of cysteine-rich receptor-like kinases (CRKs) from arabidopsis and 12 other plant species. The central string of circles represents arabidopsis CRKs. Colored circles indicate the 41 typical CRKs, three colored open circles indicate the cytoplasm-localized CRKs (CRK43, CRK44, and CRK45), and two black open circles represent a truncated CRK9 and a pseudogene CRK35. The six circles with diamonds around the central circle show the reported functions of CRKs, including regulation of growth and development, modulation of signaling pathways, hormone response, pathogen defense, stress tolerance, and adjustment of various physiological and cellular processes. The gray lines linking the colored circles or plant species with the diamonds on the six circles indicate the regulatory roles of CRKs from arabidopsis and 12 other plant species. Abbreviations: ABA, abscisic acid; arabidopsis, Arabidopsis thaliana: B. napus, Brassica napus; C. annuum, Capsicum annuum; C. sativus, Cucumis sativus; ET, ethylene; G. barbadense, Gossypium barbadense; G. max, Glycine max; H. vulgare, Hordeum vulgare; M. truncatula, Medicago truncatula; MAPK, mitogen-activated protein kinase; MeJA, methyl jasmonate; O. sativa, Oryza sativa; PCD, programmed cell death; PRs, pathogen-related genes; Pst, Pseudomonas syringae pv. tomato; P. tenuiflora, Puccinellia tenuiflora; P. vulgaris, Phaseolus vulgaris; ROS, reactive oxygen species; SA, salicylic acid; S. oleracea, Spinacia oleracea. CRKs belong to a DUF26-containing protein family with distinct domains CRKs contain an extracellular domain, a transmembrane domain (TMD), and an intracellular serine/threonine kinase domain (the classic RLK structure) [24]. The extracellular domain is composed of one to four DUF26 (domain of unknown function 26) motifs [1,22]. Most CRKs contain 2 Trends in Plant Science, Month 2023, Vol. xx, No. xx Trends in Plant Science two DUF26s in the extracellular region, whereas CRKs from Selaginella uniquely have a single DUF26, and a few CRKs from eudicots contain three to four DUF26 domains (Figure 2B). In arabidopsis, most CRKs (41 out of 44) have two DUF26s [22,25]. CRK43, CRK44, and CRK45 have no DUF26 or TMD. They are localized in the cytoplasm and may physically interact with other plasma membrane (PM)-localized CRKs [22]. In addition to CRKs, DUF26 is also a crucial domain in cysteine-rich receptor-like secreted proteins (CRRSPs) and plasmodesmata-localized proteins (PDLPs) (Figure 2B). CRKs, CRRSPs, and PDLPs form a large DUF26-containing protein family, and their phylogeny is highly intermixed [1] (Figure 2C). All DUF26-containing proteins are divided into two distinct groups: a basal group and a variable group [1]. The basal group is more conserved and includes Selaginella CRKs, a monophyletic group of CRKs from gymnosperms and angiosperms, and spruce-specific CRKs. The variable group is less conserved and includes angiosperm variable CRKs (i.e., two eudicot-specific subgroups and one monocot-specific subgroup) (Figure 2C). The CRKs might originate from fusing a single DUF26-containing CRRSP with LRR_clade_3 RLKs containing only TMD and a kinase domain in a common ancestor of vascular plants [1,26]. A CRRSP Gnk2 from ginkgo (Ginkgo biloba) and two maize (Zea mays) proteins (AFP1 and AFP2) can bind to mannose for defense against fungal pathogens [27,28], whereas PDLPs are crucial for cell-to-cell trafficking [29,30], callose deposition [31], and pathogen response [32]. In addition, the wheat peptide harboring two DUF26s, TaCRK3, has direct antifungal activity and inhibits mycelial growth of Rhizoctonia cerealis in the culture medium [33]. This implies that plant DUF26-containing proteins are crucial for the response to fungal pathogens, but the extracellular ligands and precise biochemical functions of DUF26 have not been elucidated [1]. The Cys residues form inter/intramolecular disulfide bonds for protein structural stability [1,34], and probably serve as switches that modulate the functions of some CRKs [35,36]. The conserved Cys residues in each DUF26 are usually located in the C-X8-C-X2-C motif [22] (Figure 2D), but their precise roles in modulating CRK structure and activity are not entirely clear. DUF26 Cys residues have long been proposed as targets for ROS sensing or redox regulation [22,37]. Single Cys-to-Ala mutations of AtCRK28 DUF26 (i.e., C99A, C127A, C214A, or C242A) completely abolish AtCRK28-mediated cell death in Nicotiana benthamiana, even though protein stability is not affected [38] (Figure 2E). In addition, hypersensitive cell death lesions and ROS production in arabidopsis are enhanced in CRK36-overexpressing plants but not in CRK36C76/85/88/100/119A- or CRK36C200/209/212/237A- overexpressing plants when exposed to either the necrotrophic pathogen Alternaria brassicicola or avirulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) AvrRpm1 [39]. However, the crystallographic structure of the extracellular regions of two proteins (PDLP5 and PDLP8) that are closely related to CRKs shows that all Cys residues for disulfide bond formation are not solvent-exposed but are buried inside the lectin fold [1]. This implies that the extracellular region in CRKs could form a lectin structure for signal perception. Therefore, studies on Cys exchanges with other amino acids, such as Ala [38,39], may not provide direct evidence for redox regulation and might instead reflect changes in protein folding and conformation [1]. Moreover, it is unclear whether Cys mutations in CRK28 and CRK36 affect protein subcellular localization [38,39]. Additional experiments to clarify the functions of the Cys residues in CRKs are needed. The TMD affects CRK subcellular localization. Most CRKs are localized in the PM, whereas wheat TaCRK2 and barley HvCRK1 are localized to the endoplasmic reticulum (ER) [19,40]. It is speculated that proteins with a shorter hydrophobic TMD localize to the ER, whereas proteins with a longer TMD associate with the PM [41]. The TMDs of HvCRK1 and TaCRK2 consist of 17 Trends in Plant Science, Month 2023, Vol. xx, No. xx 3 Trends in Plant Science Selaginella sdCRK Selaginella sdCRRSP Selaginella ddCRK sdCRRSP bCRK-II bCRK-I Spruce-specific CRK/CRRSP PDLP-I (C) (A) 3 4 5 Basal (α) group 1 Variable (β) group PDLP-II Amborella vCRK Amborella CRRSP Eudicot CRRSP Monocot CRRSP Monocot vCRK Eudicot vCRK (D) SP (B) sdCRK CRK_I DUF26 C C C C C-X8-C-X2-C (E) TMD DUF26 C C C C qdCRK Kinase domain D C-X8-C-X2-C ATP binding Active site DUF26 DUF26 CRK_II tdCRK K Asp Disulfide bond Lys CRCK Extracellular domain in CRK2 PDLP sdCRRSP ddCRRSP qdCRRSP Cell membrane C242A Kinase domain in CRK2 C100A C237A C127A C102A C217A C99A C214A C90A C205A C119A C212A CRK28 C209A C200A C88A C85A C76A CRK36 Trends in Plant Science (See figure legend at the bottom of the next page.) 4 Trends in Plant Science, Month 2023, Vol. xx, No. xx Trends in Plant Science and 19 amino acids, respectively, which are less than the 23 amino acids in the TMD of PMlocalized AtCRK6 [19,40]. Moreover, the specific amino acid composition of TMD is also implicated in determining protein localization [42]. When the highly hydrophobic LVL motif in the TMD was replaced by the less hydrophobic AAA, the localization of PDLP1a changed from the PM to the ER [42]. HvCRK1 and TaCRK2 contain the less hydrophobic AAA and YLW motifs at the TMD C terminus, whereas the LVG in AtCRK6 is more hydrophobic [19,40]. In addition, the R/K-rich motif following the TMD is an ER localization signal [43]. The RRLR motif in HvCRK1 and the RKAR motif in TaCRK2 adjacent to the TMDs are predicted to be a noncanonical arginine-based ER retention signal for directing protein to the ER [19,40]. It has been demonstrated that mutations in the TMD hydrophobic YLW (Y323V) and RKAR motifs (R326A/R329A and R326A/K327A/R329A) affect TaCRK2 localization to the ER [40]. The typical protein kinase domain has 11 conserved kinase subdomains [44]. The conserved Lys in subdomain II for ATP binding and Asp in subdomain VII for catalysis are crucial for CRK activity [45] (Figure 2D). Mutation of the conserved Lys to Glu (CRK2K353E ) or Asp to Asn (CRK2D450N) ablates the kinase activity of CRK2 in vitro, but these mutations do not alter CRK2 stability and subcellular localization [6,45]. Expression of these kinase-inactive CRK2 variants in the crk2 mutant does not restore the growth defects of the mutant [45]. Similarly, substitution of the conserved K by E in CRK36 (CRK36 K386E ) or by N in CRK28 (CRK28K377N) attenuates their functions in regulating stomatal closure, cell death, and growth and development [38,39]. How other amino acid residues in the subdomains affect CRK functions remains unclear. In summary, although the domain structures of CRKs have been well defined, our understanding of their functional diversity is far from complete. The TMD domain and signal peptides are certainly relevant to the subcellular localization of CRKs. Based on limited information [1,28], the lectin fold may directly bind to and sense different ligands/signals, thereby playing a role in different pathways and processes. The development of AlphaFold [46] provides a good opportunity to predict the conformations of more CRKs and to better understand their functions. CRKs function in plant immunity The different, coordinated, and redundant functions of CRKs in plant immune responses have mainly been studied in the model plant arabidopsis using reverse genetics and phenotyping [22,38,45,47]. Most AtCRK genes are clustered on chromosome 4, which may facilitate the adaptive evolution of novel specificity, subfunctionalization, or coordinated gene expression [22,38]. Because loss-of-function mutants of single CRK genes have inconspicuous phenotypes, CRK higher-order mutants and CRK-overexpressing lines are used for functional characterization [34,38,39,47–51]. Owing to the lack of genetic resources and stable transformation systems, CRKs in several crops have been characterized using various transient expression systems. For instance, transient-induced gene silencing (TIGS) analysis indicates that barley HvCRK1 (an Figure 2. Chromosomal distribution, domain composition, evolution relationship, and 3D structure of cysteine-rich receptor-like kinases (CRKs). (A) Chromosomal distribution of Arabidopsis thaliana CRK genes. The chromosome numbers are indicated at the top of the columns. Gray lines indicate segment- and tandem-duplicated genes. (B) Domain composition of plant proteins containing DUF26. The DUF26 domain is shown in green, the transmembrane domain (TMD) in blue, and the kinase domain in orange/pink); sd (single domain), dd (double domain), td (triple domain), and qd (quadruple domain) refer to the number of DUF26 domains. (C) Sketch map of the evolutionary relationship between DUF26-containing CRRSPs, CRKs, and PDLPs. The color and thickness of the lines indicate the different families and relative protein numbers, respectively. The triangles, spheres, and diamonds represent different CRRSPs, CRKs, and PDLPs, respectively. (D) Domain composition of CRK showing cysteine sites, ATP-binding sites, and the active site. (E) General domain architecture and 3D structure of CRKs. The red arrows indicate the disulfide bonds in CRK2. The conserved ATP-binding Lys residues and Asp residues in the catalytic domain are crucial for the kinase activity of CRK2. When Cys residues are mutated to Ala, the disulfide bonds are disrupted in CRK28 and CRK36. Abbreviations: CRRSPs, cysteine-rich receptor-like secreted proteins; DUF, domain of unknown function; PDLPs, plasmodesmata-localized proteins; SP, signal peptide. Trends in Plant Science, Month 2023, Vol. xx, No. xx 5 Trends in Plant Science AtCRK6 homolog) is a crucial negative regulator for defense against powdery mildew pathogen Blumeria graminis f. sp. hordei (Bgh) [19]. In addition, virus-induced gene silencing (VIGS)based analyses show that four wheat CRKs (i.e., TaCRK2, TaCRK3, TaCRK7A, and TaCRK10), pepper CaCRK5, and cotton GbCRK18 are required for plant defense against diverse pathogens [8,11,13,33,40,52]. Furthermore, quantitative trait locus (QTL) mapping reveals that two cucumber CsCRKs (Csa1M064780 and Csa1M064790) [12], wheat TaCRK6 (TaStb16q) [53], and several CRK candidates in oilseed rape [18] may play potential roles in the defense response to powdery mildew, Septoria tritici blotch, and blackleg. However, the molecular mechanisms underlying CRK functions in plant immune responses remain largely unexplored. Plants have developed pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) in their defense against microbial infections. PTI is triggered by pathogen-/microbe-associated molecular patterns (PAMPs/MAMPs) which are recognized by surface-localized pattern-recognition receptors (PRRs). ETI is initiated by plant resistance (R) proteins after recognizing pathogen effectors [54–56]. The characteristics of PTI include a series of physiological and cellular structural changes, such as rapid Ca2+ influx, apoplastic ROS burst, activation of Ca2+-dependent protein kinase (CPK) and MAPK cascades, alteration of defense hormone networks, callose deposition, stomatal closure, and extensive transcriptional, translational, and metabolic reprogramming for defense [57–60]. By contrast, ETI usually induces a stronger and more prolonged immune response than PTI, often leading to a hypersensitive response (HR) associated with PCD at the infection site to restrict pathogen spread [60]. HR can induce a systemic acquired resistance (SAR) which exhibits plant-wide, long-lasting, and broad-spectrum resistance to subsequent pathogen infection [61]. Historically, PTI and ETI have been regarded as binary or zig-zag processes [62,63]. In reality, PTI and ETI share central hubs of signaling including ROS burst, MAPK cascades, and activation of PR genes [64,65]. Accumulating evidence indicates that CRKs are involved in regulating both PTI and ETI responses. CRKs associate with PRRs in the PTI process PRRs are pivotal cell-surface receptors that trigger PTI responses upon recognition of PAMPs [66]. The LRR-RLK FLAGELLIN-SENSITIVE 2 (FLS2) is a well-characterized PRR, and it can recognize the conserved bacterial flagellin peptide flg22 [67]. Upon flg22 perception, FLS2 recruits another LRR-RLK BRASSINOSTEROID INSENSITIVE 1 (BRI1)-associated kinase 1 (BAK1, also called SERK3), and they form a heterodimer [68–71]. Malectin-like LRR-RLK IMPAIRED OOMYCETE SUSCEPTIBILITY 1 (IOS1) can promote the FLS2–BAK1 association [72], which subsequently phosphorylates and activates their downstream targets such as receptor-like cytoplasmic kinases (RLCKs) including BOTRYTIS-INDUCED KINASE 1 (BIK1) [39,73,74] (Figure 3A). PRRs constitute a central hub of PM-localized multiprotein complexes for timely PTI responses [34,75]. A subset of CRKs interacts with the PRR FLS2. They are proposed to be potential components of the PRR complex for recognizing flg22 [34,38,39]. Specifically, PM-localized CRK4, CRK6, and CRK36 associate with the PRR FLS2 in a ligand flg22-independent manner, and overexpression of these CRKs enhances flg22-triggered ROS production [34] (Figure 3A). CRK36 also directly interacts with BIK1 to enhance flg22-triggered BIK1 phosphorylation [39] (Figure 3A). In addition, CRK28 is associated with BAK1 or FLS2 in a ligand-independent manner which is also independent of the kinase activity of CRK28 [38]. Importantly, the CRK28–FLS2– BAK1 module forms a PRR immune complex in a flg22-dependent manner [38] (Figure 3A). Therefore, CRKs are implicated in contributing to the structural stability and phosphorylation cascade of the PRR FLS2 complex. However, whether other CRKs form PRR complexes and the nature of the downstream signaling partners and the dynamics of the complexes remain to be investigated. Notably, CRK28 self-associates to form homodimers, or associates with its closely related CRK29 to form heterodimers [38] (Figure 3A). It will also be interesting to investigate 6 Trends in Plant Science, Month 2023, Vol. xx, No. xx Trends in Plant Science (A) flg22 ROS FLS2 BAK1 IOS1 FLS2 CRK CRK6/16 Ca2+ BAK1 /19/20 channel CRK2 28/29 CRK5/22 /22/23 /24/25 Callose deposits CRK4/6 /29/32 CRK28 CRK28 CRK36 Callose RBOHD CRK3/ 13/31 deposits 343 339 347 P P 163 Ca 148 BIK1 39 133 P 2+ P BIK1 703 ˛ CALS1 P P MP3K [Ca2+] BIK1 flg22 ROS P MP2K P CPK5 ˛ ˛ P MPK3/6 Pattern-triggered immunity (B) SA Pst DC3000 P. triticina SA Pst DC3000 A. brassicicola X. oryzae pv. oryzae Fumonisin B1 Pst DC3000 AvrRpm1 V. dahliae toxins ROS BAK1 TaCRK2 [Ca2+] CRK CRK28 CRK4 19/20 CRK13 Glycosylation N* ER CRK 5/22 FHA PP2C 11/70 P ? FHA NPR1 NPR1 OsNH1 PR1 OsCRK10 TGA2/6 TGA2.1 P SA PR genes PR1a/1b/2/10 ? CRK10 WRKY18 W-box TATA-box PR genes PR genes PR genes PR1/5 PR1/2 AIG1 PR1/2/5 Programed cell death CRK 6/11 PP2C 11/70 P ? P SA CRK45 WRKY70 CRK10 CRK5 SA W-box PR genes TaPR1/2/5 OsCRK10 CRK45 ˛ ˛ ICSI EDS5 ? NahG Golgi CRK36 PR gene PR1 Resistance Trends in Plant Science Figure 3. Schematic model for cysteine-rich receptor-like kinase (CRK) function in plant immunity. (A) CRK interacts with other RLKs to form PRR immune complex upon pattern-triggered immunity (PTI). Upon flg22 perception, CRK36 enhances the function of the FLS2–BAK1–BIK1 module to phosphorylate the N terminus of RBOHD for apoplastic ROS production. The CRK28–FLS2–BAK1 module works as a PRR immune complex in a flg22-dependent manner to trigger ROS accumulation. CRK28 can associate with another CRK28 or CRK29 to form homodimers or heterodimers, respectively. CRK2 directly phosphorylates the C terminus of RBOHD at Ser703 for ROS production. Ca2+ influx into the cytosol and the Ca2+-dependent CPK activation is important for phosphorylating the N terminus of RBOHD. In addition, CRK2 mediates the inhibition of the MAPK cascade and callose deposition, possibly via CALS1. Three CRKs (CRK3, CRK13, and CRK31) induce apoplastic ROS elevation, whereas the other ten CRKs probably decrease ROS production. (B) CRK regulation of programmed cell death to defend against pathogens. CRK5, CRK6, CRK10, and CRK11 are induced by Pst DC3000 and SA. The W-box sequence region of CRK10 is recognized by WRKY18, and binding activity is induced by SA. CRK5 and CRK10 interact with PP2C70, PP2C11, and FHA, which probably alter CRK kinase activity by dephosphorylation. CRK19, CRK20, and N-glycosylated CRK4 are also induced by SA and pathogen Pst DC3000. N-glycosylated CRK28 interacts with BAK1 on PM for sensing flg22 and mediating programmed cell death. Wheat TaCRK2 localized in the ER and PM positively regulates the HR cell death by modulating PR genes after Puccinia triticina infection. CRK13 promotes SA accumulation (Figure legend continued at the bottom of the next page.) Trends in Plant Science, Month 2023, Vol. xx, No. xx 7 Trends in Plant Science whether the dimerization of CRKs facilitates PRR complex formation for the rapid recruitment of downstream signaling components. CRKs modulate the shared signaling hubs of PTI and ETI ROS, as important signaling molecules, regulate plant development and stress responses. In plants, the PM-localized NADPH oxidase RBOH family functions in apoplastic ROS production. Arabidopsis RBOHD is a key player in the response to pathogen infection. It has been well characterized to be phosphorylated by CRK2 [45] and several other kinases such as BIK1 [73,74] and CPKs [76]. For example, Ser39, Ser339, Ser343, and Ser347 of RBOHD are phosphorylated by BIK1 [73,74], whereas Ser133, Ser148, Ser163, and Ser347 are phosphorylated by CPK5 [76] (Figure 3A). CRK2 regulates RBOHD activity by interacting with the cytosolic N-terminal or C-terminal regions of RBOHD [45] (Figure 3A). CRK2 phosphorylates N-terminal Ser8 and Ser39 and C-terminal Ser611, Ser703, and Ser862 residues of RBOHD [45]. Notably, only two of the five CRK2 phosphorylation sites in RBOHD (Ser703 and Ser862) are crucial for ROS production. Specifically, phosphorylation of Ser703 and Ser862 by CRK2 positively and negatively regulates RBOHD activity, respectively [45]. Phosphoproteomic analysis has revealed that phosphorylation of Ser703 and Ser39 in RBOHD is enhanced in arabidopsis after flg22 treatment for 5 minutes [45]. Further genetic analyses have shown that CRK2 mediates flg22-induced phosphorylation at Ser703 of RBOHD (Figure 3A) [6,45]. ROS production is decreased in both the crk2 and bik1 mutants, implying that CRK2 and BIK1 may synergistically regulate RBOHD phosphorylation for apoplastic ROS production [45] (Figure 3A). In addition to CRK2, CRK36 interacts with BIK1 and FLS2, which is also required for RBOH-mediated ROS production [34,39] (Figure 3A). RBOHD/ F-mediated ROS production, flg22-induced stomatal closure, and resistance to Pst DC3000 are enhanced in CRK36-overexpressing plants but are compromised in the crk36 mutant [39]. The CRK36–BIK1–RBOHD/F module is proposed to form a feedback activation loop that mediates rapid and transient ROS production during stomatal immunity. Similarly to CRK36, overexpression of CRK4 and CRK6 also primes ROS production upon flg22-triggered PTI, but the regulatory mechanism is unclear [34] (Figure 3A). In addition, CRK28-mediated ROS accumulation is specifically induced by treatment with flg22 but not with chitin. This suggests that CRK28 enhances the ROS burst in response to specific pathogens [38]. CRK-mediated ROS production via RBOHD phosphorylation is a finely tuned process. Only CRK2 has been shown to phosphorylate RBOHD [45], but whether RBOHD can be phosphorylated by other CRKs requires further study. In some crk mutants, ROS production is obviously perturbed under flg22 treatment, with increases in ROS production in 11 mutants (i.e., crk6, crk16, crk19-2, crk20, crk22, crk23-1, crk23-2, crk24, crk25, crk29, and crk32) and decreases in four mutants (i.e., crk2, crk3, crk13, and crk31) [22] (Figure 3A). The reverse genetics data from the aforementioned 11 mutants indicate that these CRKs may be negative regulators, by inducing ICS1 and PR genes to cope with pathogens. Cytoplasm-localized CRK45 interacts with PM-localized CRK36, thus positively regulating plant resistance to pathogens and SA. Pathogen-induced SA accumulation promotes NPR1 translocation, triggering WRKY70 expression and subsequently enhancing CRK45 transcription. Abbreviations: A. brassicicola, Alternaria brassicicola; AIG1, AvrRpt2-induced gene 1; BAK1, BRI1-associated receptor kinase 1; BIK1, Botrytis-induced kinase 1; CALS1, callose synthase 1; CPK5, calcium-dependent protein kinase 5; EDS5, enhanced disease susceptibility 5; ER, endoplasmic reticulum; FHA, FHA domain-containing protein; flg22, bacterial flagellin peptide 22; FLS2, flagellin-sensing 2; Golgi, Golgi apparatus; HR, hypersensitive response; ICS1, isochorismate synthase 1; IOS1, impaired oomycete susceptibility 1; MPK, mitogen-activated protein kinase; MP2K, mitogen-activated protein kinase kinase; MP3K, mitogen-activated protein kinase kinase kinase; NahG, salicylate hydroxylase; NH1, NPR1 homolog 1; NPR1, nonexpressor of PR genes 1; P, phosphorylation; PM, plasma membrane; PP2C, protein phosphatase 2C; PR, pathogenesisrelated protein; PRR, pattern-recognition receptor; Pst, Pseudomonas syringae pv. tomato; RBOHD, respiratory burst oxidase homolog protein D; RLK, receptor-like kinase; ROS, reactive oxygen species; SA, salicylic acid; STT3a, oligosaccharyl transferase subunit STT3A; TGA2.1, transcription factor TGA2.1; WRKY, WRKY DNA-binding protein; X. oryzae pv. oryzae, Xanthomonas oryzae pv. oryzae. 8 Trends in Plant Science, Month 2023, Vol. xx, No. xx Trends in Plant Science whereas the data from the other four mutants indicate that the CRKs may be positive regulators. Notably, crk5 and crk28 mutants exhibit normal ROS production but severe disease symptoms after Pst DC3000 infection, whereas crk23 elevates flg22-induced ROS levels but does not improve defense against Pst DC3000. Enigmatically, crk20 and crk29 plants with elevated ROS levels are more susceptible to Pst DC3000 compared to the wild type (WT). Clearly, ROS levels in the crk mutants do not predict disease resistance. Ca2+ is a transient cellular messenger, and an increase in cytoplasm Ca2+ concentration ([Ca2+]cyt) is a prominent trigger for initiating downstream immune responses [77]. [Ca2+]cyt is rapidly induced in WT plants but is decreased in the crk2 mutant [45]. This indicates that CRK2 is an essential component for controlling Ca2+ influx during the immune response (Figure 3A). In turn, CRK is probably regulated by calcium signals. The induction of TaCRK2 transcription by Puccinia triticina in wheat is inhibited following treatment with the extracellular Ca2+ chelator EGTA [ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid] [40]. This implies that TaCRK2 functions in a Ca2+-dependent manner in wheat defense against pathogens. MAPK cascades are highly conserved in modulating multiple defense responses. MAPK activation is the earliest signal, even after sensing PAMPs in PTI and pathogen effectors in ETI [78]. CRK2 might be a negative regulator of the MAPK cascade because flg22-induced MAPK activation is enhanced in the crk2 mutant [45]. Thus, CRK2 could be involved in fine-tuning the PAMPinduced MAPK signaling pathway (Figure 3A). However, flg22-induced expression of CRK28 does not significantly affect MAPK activation [38] (Figure 3A). In addition, CRK5 and CRK22 are involved in the activation of MPK3 and MPK6 to cope with Verticillium dahliae (Vd) toxins [79] (Figure 3A). This suggests that different CRKs differentially modulate MAPK signaling during the plant immune response. Callose deposition between the PM and cell wall in epidermal cells, plasmodesmata, and vascular tissue can restrict pathogen ingression and spread [80]. Callose deposition is increased in CRK36-overexpressing plants irrespective of Pst DC3000 hrcC− and flg22 treatments but is decreased in crk36-2 mutants [34,39]. Similarly, callose deposition is increased in CRK5- and CRK22-overexpressing plants but decreased in their mutants upon Vd toxin treatment [79] (Figure 3A). By contrast, CRK4 or CRK6 overexpression has no effect on PTI-mediated callose deposition [34]. Notably, early callose deposition is enhanced in crk2 mutants after 30 min of flg22 treatment, whereas callose deposits are comparable in the WT and crk2 mutants at 12 h after flg22 treatment [45]. These results imply that CRK-mediated callose deposition is crucial for the late PTI process. Although it is reported that CRK2 interacts with callose synthase 1 (CALS1) under salt stress and phosphorylates it in vitro [6], the role of salt stress-triggered callose deposition and the molecular mechanism of CRK-mediated callose deposition are not completely understood. Stomatal movement is modulated by diverse factors such as phytohormones [e.g., abscisic acid (ABA), methyl jasmonate (MeJA), brassinosteroid, salicylic acid (SA), cytokinin, and auxin], ROS, Ca2+ signaling, and osmotic homeostasis [22,81]. As an important part of PTI, stomatal closure prevents water loss and limits pathogen entry [82,83], but prolonged stomatal closure creates aqueous apoplasts, thus promoting colonization by pathogens [84,85]. Different members of the CRK family act as functionally diverse and/or specific regulators of stomatal immunity [22] (Figure S2 in the supplemental information online). Flg22-triggered stomatal closure is compromised in the crk2, crk5, crk10-2, crk17, crk20, and crk28 mutants, resulting in increased susceptibility to Pst DC3000 [22,45]. Chitin-triggered stomatal closure is also retarded in the crk2, crk6, crk10-2, crk10-4, crk12, and crk19-2 mutants, while only the crk2 and crk10-2 mutants exhibit Trends in Plant Science, Month 2023, Vol. xx, No. xx 9 Trends in Plant Science impaired stomatal closure in response to flg22 and chitin [22] (Figure S2). In addition, CRK6 overexpression induces constitutive stomatal closure, whereas CRK4 and CRK36 overexpression counteracts Pst DC3000-mediated stomatal reopening in a coronatine (COR)-dependent manner [34,86] (Figure S2). Pst-induced stomatal reopening is completely abolished in CRK36overexpressing plants but is more pronounced in the crk36-2 mutant (Figure S2). Importantly, CRK36 phosphorylates BIK1 [39], and BIK1 subsequently phosphorylates RBOHD/F to trigger apoplastic ROS production [73,74] (Figure S3 in the supplemental information online). In the bik1 and rbohD/F mutant backgrounds, CRK36-mediated stomatal closure and prevention of reopening are impaired in response to flg22 and Pst DC3000 treatments (Figure S2). These data suggest that CRK36 acts through the BIK1–RBOHD/F signaling module to positively regulate stomatal immunity [39]. Various phytohormones such as SA, MeJA, ethylene, and ABA play synergistic or antagonistic roles in plant pathogen immunity. Generally, SA signaling induces defense against biotrophic and hemibiotrophic pathogens, whereas MeJA and ethylene cooperatively activate resistance against necrotrophic pathogens [87]. Although ABA is known to function mainly in abiotic stress, the enhancement of ABA signaling correlates with susceptibility to disease caused by several plant pathogens [88]. The transcription levels of several CRKs in arabidopsis, wheat, and pepper are changed in response to exogenous SA [11,37,49–51,89], MeJA [8], ethylene [33], and ABA [90] (Figure 3B and Figure S3). In arabidopsis, nine CRK transcripts (i.e., CRK 4, 5, 6, 10, 11, 13, 19, 20, and 45) are induced upon SA treatment and pathogen infection [48–51,89] (Figure 3B). They all have a cluster of W-box elements in their promoter regions for binding WRKY transcription factors, and the binding of WRKYs to the W-box in the CRK10 promoter is induced by SA [89] (Figure 3B). Importantly, SA-induced expression of CRK5, CRK13, and CRK45 can promote downstream PR1 gene expression [49–51], whereas overexpression of CRK4, 5, 13, 19, 20, and 22 triggers rapid PCD [48,51,79] (Figure 3B). Compared to arabidopsis, wheat has a larger CRK family with 170 members. Most TaCRKs exhibit differential temporal expression patterns in response to different pathogens and phytohormones [8,9,33,52,90] (Figure S3). TaCRK1 transcription is induced by ABA at 3 h and by MeJA at 12–24 h post-treatment, but is decreased by MeJA at 1–6 h post-treatment and is also downregulated after ethylene and SA treatments. This clearly shows the temporal dynamics of TaCRK1 regulation in response to different hormones [90]. By contrast, TaCRK3 is significantly induced by ethylene treatment, whereas TaCRK3 silencing decreases the expression of ethylene biosynthesis and signaling genes in wheat, such as ACO2 encoding an ethylene biosynthesis enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase [33] (Figure S3). This suggests that TaCRK3 may act as an upstream regulator of ethylene biosynthesis and signaling, and that the ethylene pathway in turn induces TaCRK3 expression in a feedback regulation manner [33]. Furthermore, TaCRK7A is significantly induced after JA treatment, whereas in TaCRK7A knockdown wheat lines several JA-responsive genes are downregulated, such as PR2, TaGluD, and TaChit1/ 3/4 [8] (Figure S3). This implies that the TaCRK7A regulatory loop triggered by JA is similar to that of TaCRK3 by ethylene. In addition, TaCRK10 is induced by SA, but is downregulated after ABA, MeJA, and ethylene treatments [52] (Figure S3). Silencing of TaCRK10 attenuates the disease resistance of wheat cultivar 'XY6', whereas overexpression of TaCRK10 in susceptible wheat variety 'Fielder' increases resistance by inducing the expression of TaPR1 and TaPR2 genes in the SA signaling pathway [52]. Upon Puccinia striiformis inoculation and high-temperature stress, TaCRK10 physically interacts with and phosphorylates TaH2A.1, and the phosphorylated TaH2A.1 transfers into the nucleus for regulating immune-related gene expression (Figure S3). Histone modification has been reported to be involved in SA signal transduction in response to diverse pathogens [91], and silencing TaH2A.1 can suppress wheat resistance [52]. Interestingly, 10 Trends in Plant Science, Month 2023, Vol. xx, No. xx Trends in Plant Science CaCRK5 from pepper is also induced by SA, and is directly regulated by homeodomain zipper I protein CaHDZ27. Pepper CaCRK5 is involved in regulating SA-mediated signaling and the expression of several downstream genes involved in defense against Ralstonia solanacearum [11]. In addition, CaCRK5 can form a heterodimer with CaCRK6 on PM, but the mechanisms of this dimerization and its biological roles are not clear [11] (Figure S3). It is also not known whether TaCRK3 and TaCRK7A regulate ethylene and JA signaling, respectively, through epigenetic mechanisms. CRKs regulate ETI PCD to defend against pathogens In plants, the PCD process is an inherent part of regular development and responses to biotic and abiotic stress [92]. Pathogen-triggered PCD (pPCD) occurs at infection sites and surrounding areas in host plants, and it is mediated by various phytohormones such as ethylene [93], JA [94], and SA [95,96]. Importantly, calcium is proposed to be both a positive regulator for the timely triggering of pPCD [97] and a negative regulator for suppressing SA-dependent defense [98]; the positive feedback loop between SA and ROS is also assumed to be a key pPCD trigger [99]. Several arabidopsis CRKs and rice OsCRK10 are involved in SA-mediated pPCD [10,47–49,51,79]. In CRK13- and CRK45 (ARCK1)-overexpressing plants, the expression of multiple SA-related genes is induced, including the SA biosynthesis gene ICS1, the SA transporter gene EDS5, and several SA-responsive PR genes [49,51]. In addition, pathogen-induced SA accumulation promotes translocation of the transcription activator NPR1 from the cytoplasm to the nucleus where it triggers WRKY70 gene expression and subsequently enhances CRK45 transcription (Figure 3B) because CRK45 expression is decreased in npr1 and wrky70 mutants [49] but increased in a sni1 (NPR1 suppressor) mutant upon pathogen infection [100]. This is another example of a positive feedback loop. Similarly, Vd toxin-induced CRK5 and CRK22 positively regulate SA biosynthesis and then modulate NPR1 expression. Subsequently, NPR1 is recruited by transcription factor TGA to induce the expression of PR genes [79] (Figure 3B). Consistent with this, transcription factor OsTGA2.1 interacts with OsNH1 (an NPR1 homolog) to trigger downstream OsCRK10 expression, which induces the expression of multiple PR genes for the enhancement of rice resistance to infection by Xanthomonas oryzae pv. oryzae (Xoo) [10] (Figure 3B). Moreover, in the loss-of-function mutant of SA biosynthesis, dexamethasone (DEX)-induced expression of DEX:CRK13 delays the phenotype of HR-like cell death [51]. Similarly, NahG transgenic plants (SA-deficient) exhibit obvious reductions of CRK45 expression and disease symptoms after pathogen infection [49] (Figure 3B). This implies that pathogen-induced SA accumulation exhibits positive feedback that enhances CRK-induced PCD. PCD is closely correlated with pathogen growth and spread [39]. CRK36-overexpressing plants exhibit dramatic ROS burst and cell death symptoms after infection by the necrotrophic pathogen A. brassicicola, avirulent Pst DC3000 AvrRpm1 and AvrRpt2, and PCD-eliciting fungal toxin fumonisin B1, in contrast to the crk36 mutant [39] (Figure 3B). Enhanced PCD facilitates the growth of A. brassicicola. Conversely, after infection by virulent Pst DC3000, disease symptoms and ROS accumulation are reduced in CRK36-overexpressing plants but become severe in crk36-2 seedlings [39]. This indicates that CRK36-induced PR gene expression and PCD positively restrict virulent pathogen growth [39] (Figure 3B). Interestingly, TaCRK2 also positively regulates HR by modulating several PR genes to cope with P. triticina infection, but whether this is SA-mediated is unknown [40] (Figure 3B). Notably, some CRK-overexpressing plants exhibit 'dose-dependent' phenotypes of pathogen tolerance. The highly DEX-inducible and moderately constitutive overexpression of CRKs shows distinct functions in plant growth and stress tolerance [50,51]. For instance, 5 μM DEXTrends in Plant Science, Month 2023, Vol. xx, No. xx 11 Trends in Plant Science induced high expression of DEX:CRK13 triggers rapid cell death, but 0.05 μM DEX-induced moderate expression of DEX:CRK13 enhances arabidopsis resistance to Pst DC3000 [51]. In addition, DEX-induced high expression of DEX:CRK5 triggers HR-like cell death which is associated with H2O2 accumulation and nuclear DNA fragmentation, but constitutive 35S:CRK5 overexpression promotes leaf growth and resistance to Pst DC3000 by quickly inducing PR1 genes [50]. This indicates that the manner and/or level of CRK induction or activation in transgenic plants can have different effects on the tradeoff between leaf growth/disease resistance and the PCD process. The ATP-binding site (K) and the potential N-glycosylation site (N) in CRK4, CRK5, and CRK28 are crucial for their kinase activities or subcellular localization that are necessary for regulating PCD [38,50,101]. Kinase-inactive DEX:CRK5K368E transgenic plants exhibit no cell death phenotype [50]. In addition, N-glycosylation of N181 in CRK4 through STT3a in the ER is crucial for its protein stability [101]. Transient expression of CRK4N181Q in N. benthamiana leaves leads to reduced PCD intensity compared to WT CRK4 [101]. Moreover, N-glycosylation in the ER and Golgi apparatus and K377-dependent kinase activity are required for CRK28 modulation of PCD [38]. However, the exact N-glycosylation sites of CRK28 and post-translational modification (PTM) regulatory mechanisms remain to be revealed. Interestingly, two rice OsCRK10 single amino acid substitution (V429 to I/L429) mutants, LIL1 and als1, exhibit SA accumulation, ROS burst, increased expression of PR genes, and HR-like cell death in leaves [61,102], while overexpression of OsCRK10V429I induces a LIL1 lesion phenotype in 'Nipponbare' rice [61]. However, these primary phenotype studies on lesion-mimicking mutants of rice does not explain whether a single amino acid substitution affects OsCRK10 function in regulating SA and ROS signaling to cope with rice blast [61,102]. A few CRK-interacting proteins have illuminated the pathogen signal sensing and transduction to regulate PCD. CRK28 interacts with the RLK coreceptor BAK1 for mediating PCD because CRK28-induced cell death is significantly decreased in NbSerk3 (BAK1)-silenced tobacco leaves [38,103] (Figure 3B). In addition, although CRK10-overexpressing plants exhibit no inconspicuous PCD phenotypes, both CRK10 and CRK5 can physically interact with several type 2C protein phosphatases (PP2Cs) such as PP2C70, PP2C11, and an FHA domain-containing protein (At2g21530) [48] (Figure 3B). However, whether the activities of CRK5 and CRK10 are modulated through PP2C dephosphorylation requires further investigation. Interestingly, cytoplasmlocalized CRK45 has been shown to interact with PM-localized CRK36, but how their interaction senses and transduces extracellular signals remains elusive [2]. In summary, CRKs clearly play important roles in PTI and ETI processes. Some CRKs (e.g., CRK2 and CRK36) are better studied than others, but most of the work has been descriptive and correlative, and further work will be necessary to provide a mechanistic understanding. Important questions remain unanswered, such as whether CRKs function through kinase activity, why some CRKs are positive regulators whereas others are negative regulators, and how different CRK signaling pathways crosstalk. The positive feedback loops involving TaCRK3 and ethylene, TaCRK7A and JA, and CRK45 and SA deserve further study. How the loops regulate is not yet known. Considering the large and diverse CRK family and the fact that many CRKs may have redundant functions, elucidating CRK molecular networks in plant immunity seems to be a daunting task. Large-scale interacting proteomics and PTM proteomics together with reverse genetics tools may shed light in the future. CRKs are involved in abiotic stress Drought and salinity stress lead to osmotic imbalance. Plants have evolved sophisticated mechanisms for regulating stomata movement and cell-wall dynamics to cope with osmotic stress. 12 Trends in Plant Science, Month 2023, Vol. xx, No. xx Trends in Plant Science Specifically, ABA is known to play an important role in modulating stomatal closure and osmotic homeostasis [104]. Some CRKs are involved in ABA signaling and biosynthesis [2,105,106]. CRK5, together with its homolog CRK4, redundantly enhances plant drought tolerance by positively regulating the ABA sensitivity of stomatal movement [106]. Interestingly, the ABAresponsive CRK5 is modulated by a feedback and feed-forward mechanism because CRK5 expression is cooperatively inhibited by three homologous ABA-regulated transcription factors, WRKY18/40/60, and CRK5 functions upstream of ABI2 in ABA signaling [4,106,107]. In addition, cytoplasm-localized CRK45 positively regulates the expression of ABA biosynthesis genes such as 9-cis-epoxycarotenoid dioxygenases (NCED3 and NCED5), ABA deficient (ABA1 and ABA2), and abscisic aldehyde oxidase 3 (AAO3), and enhances tolerance to drought and salt stress upon germination and post-germination growth [105]. However, CRK45 also interacts with and is phosphorylated by CRK36 to negatively regulate ABA and osmotic stress signaling in postgermination growth [2]. In CRK36 RNAi plants, ABA treatment induces several ABA-responsive genes such as late embryogenesis abundant (LEA), oleosin (OLEO4), ABI4, and ABI5 [2]. This implies that CRK45 plays opposite roles under different experimental conditions such as stratification and ABA treatments [2,105]. Callose is a key regulator of plasmodesmata transport in response to osmotic and salinity stress [108]. CRK2 promotes callose deposition at plasmodesmata, thereby enhancing plant salt stress tolerance at the germination stage [6]. Salt stress induces an increase in cytosolic Ca2+ and triggers phospholipase D α1 activity, which are required for CRK2 relocalization from the PM to the nanodomain in plasmodesmata [6]. The ATP-binding K353 and the D450 in the catalytic core are essential for CRK2 activity, which phosphorylates CALS1 to promote callose deposition for the regulation of plasmodesmata permeability against osmotic stress and Na+ toxicity during salt stress [6]. In addition, our previous transcriptomic analysis revealed that 17 alkaligrass PutCRKs exhibited salinity-induced expression changes in leaves and roots under NaCl, Na 2CO 3, and NaHCO 3 treatments [21] (Figure S1). However, their regulatory mechanisms are unknown. CRKs may be involved in oxidative stresses caused by H2O2, O3, and UV light [3,6,22,37,47]. H2O2-induced extracellular ROS accumulation alters the localization pattern of CRK2, possibly causing it to form microdomains [6]. The crk2, crk5, crk40, and crk42 mutants exhibit a significant elevation of electrolyte leakage under oxidative stress conditions, and genetic complementation of crk5 rescues its hypersensitivity to UV radiation [22]. In addition, more than 25 CRKs can redundantly function in response to O3 [22,37]. For example, CRK6, CRK7, and CRK20 are induced by O3 in WT plants, but their mutants show little change in phenotype, physiology, and antioxidant gene expression [3,47]. Overall, only a small number of CRKs have been found to be involved in plant abiotic stress responses. The molecular mechanisms underlying CRK functions in plant abiotic stress have not been sufficiently studied. CRKs balance the plant stress response and growth In adverse environments, sessile plants have evolved sophisticated mechanisms to balance growth and stress responses [109]. Active growth inhibition is an adaptive strategy for facilitating plant survival under stress conditions [104]. Various CRK-mediated pathways such as ROS production [22,38,45], Ca2+ signaling [45], MAPK cascade [38,45], and ABA signaling are essential for both stress response and plant growth. Therefore, CRKs may be involved in regulating the growth–stress tolerance tradeoff. Reverse genetics data of several CRKs suggest their potential roles in the balance of growth and stress tolerance [7,45,105,106,110]. For instance, CRK2, CRK5, and CRK36 mediate callose Trends in Plant Science, Month 2023, Vol. xx, No. xx 13 Trends in Plant Science deposition of immune response, whereas their mutant phenotypes imply that they might be involved in the regulation of seed germination, seedling growth, rosette leaf size, and root development [6,34,39,45,79] (Figure 4). This indicates that CRK-mediated callose deposition may also regulate cell expansion-driven growth. Similarly, CRK28, a key regulator of plant immunity, inhibits the initiation of lateral root primordia and primary root meristems, and decreases silique length and seed number [7,38] (Figure 4). CRK28 also positively controls root hair development, rosette size, and inflorescence branches [7]. In addition, CRK45 is a modulator of ABA, osmotic, and salt stresses [2,105], and may also regulate bolting and early seedling development [105] (Figure 4). Several CRKs mediate stomatal movement in response to stress and stomata development (length and density) under normal conditions [22] (Figure S2). CRK33 is preferentially expressed in leaves and cotyledons, and regulates stomatal spacing and development by modulating the expression of several genes involved in guard cell fate such as SPEECHLESS (SPCH), TOO MANY MOUTHS (TMM), MUTE, and FAMA [110] (Figure 4). In crk33-2 and crk33-3 mutants, stomatal density and stomatal index are decreased in the leaves and cotyledons, leading to decreased stomatal conductance and transpiration, as well as enhanced water-use efficiency and drought tolerance [110]. In addition, ABA-induced stomatal closure is accelerated in the crk22, crk24, crk37, and crk46 mutants compared to the WT [22]. In detached leaves, stomatal closure is impaired in crk2, crk5, and crk31 mutants, leading to increased water loss and the rapid decrease of fresh weight, but water loss is less pronounced in the crk45 mutant compared to the WT (Figure S2). These phenomena can be rescued by complementation of the crk2, crk5, and crk45 mutations, respectively, with the corresponding WT genes [22]. CRK-mediated stomatal movement regulates the water use and the CO2 concentration in mesophyll cells, thus contributing to photosynthetic carbon fixation and energy supply. The extent to which CRK-mediated stomatal movement contributes to the balance between the stress response and growth is not known. CRK5 and CRK36 are not only necessary for the response to pathogen infection and abiotic stress (i.e., osmosis, oxidation, and salinity) but also regulate plant senescence (Figures 3 and 4). Stressinduced senescence benefits plant survival but decreases productivity [111]. CRK5 negatively regulates ethylene and H2O2 production through SA signaling in the senescence process [4]. The crk5 mutant displays smaller young seedlings, rapid cell death in cotyledons, impaired stomatal conductance, and accelerated senescence, but the CRK5 overexpressing line shows slightly larger rosettes [22] and shorter roots in young seedlings [106]. Ethylene accumulation, the expression of ERF1 and PDF1.2 in ethylene signaling pathways, and SA accumulation are induced in the crk5 mutant [4]. SA accumulation correlates with the increase of the positive SA signaling regulator WRKY53 and the decrease of the negative regulator WRKY70 which can recognize the W-box elements enriched in the CRK5 promoter region [4] (Figure 4). In addition, CRK36 is also involved in leaf senescence, which is revealed by the obvious early-senescence phenotype in CRK36overexpressing plants and delayed senescence in crk36-2 leaves [39] (Figure 4). Current data suggest that CRK5 and CRK36 may be involved in growth/senescence processes, and CRK5 seems to function by regulating stress-related hormones in a tissue-specific and temporal manner [104,112]. Clearly, the current results do not address whether the growth/senescence phenotypes represent pleiotropic effects or the direct functions of CRKs. Future studies using inducible knockdown systems will be important for determining the causal effects of CRK functions. Based on the results from a small number of CRKs, the balance between stress and growth may be modulated through CRK-mediated stomatal movement and stress hormone crosstalk. 14 Trends in Plant Science, Month 2023, Vol. xx, No. xx Trends in Plant Science Outstanding questions How do PTMs (e.g., glycosylation and phosphorylation) regulate CRK activity and functions? ERF1 PDF1.2 Is CRK dimerization/multimerization regulated by Cys redox in the DUF26 domain, and how does redox modification subsequently modulate CRK structure and function? WRKY53 70 SPCH TMM MUTE FAMA What are the extracellular ligands of CRKs during plant immune responses? What are the signaling components downstream of CRKs in response to different environmental stresses? What are the mechanisms and roles underlying the formation of complexes between CRKs and PRRs upon PAMP/MAMP perception? ABI3 5 Trends in Plant Science Figure 4. Participation of cysteine-rich receptor-like kinases (CRKs) in the regulation of plant growth and development. CRK2 induces seedling growth whereas CRK33 facilitates stomatal spacing and development. CRK5 negatively regulates ethylene and SA signaling, leading to increased seedling height and rosette leaf growth but decreased root hair growth. CRK36 promotes leaf senescence whereas CRK45 regulates the delay in bolting time. CRK28 is a repressor for the initiation of early lateral root primordia and primary root meristems induced by ABA signaling. CRK28 positively controls root hair development, growth delay, rosette size, and inflorescence branches, but reduces silique length and the number of seeds. Abbreviations: ABA, abscisic acid; ABI, abscisic acid-insensitive; ERF1, ethylene-responsive factor 1; ET, ethylene; FAMA, transcription factor FAMA; MUTE, transcription factor MUTE; PDF1.2, plant defensin 1.2; SA, salicylic acid; SPCH, Speechless; TMM, too many mouths; WRKY, WRKY DNAbinding protein. How do CRKs regulate HR cell death, and how are SA and other hormones involved in CRK-mediated HR processes? How do CRKs regulate cell division and differentiation in different tissues and organs during plant growth and development? How do different CRKs modulate the sophisticated hormone signaling networks in various stress responses and growth processes? Concluding remarks and future perspectives Tackling the functional redundancy and diversity of CRKs Plant CRKs can sense and transduce extracellular signals to fine-tune immune responses, abiotic stress tolerance, and growth and development. Based on the current knowledge summarized in this review, it is reasonable to conclude that CRKs are emerging regulators in many of these processes (see Outstanding questions). However, the clustered distribution on the chromosomes and the evolutionary redundancy of most CRKs make it difficult to accurately determine the unique function of each CRK through molecular genetics analyses in loss-of-function mutants [22]. Most studies on CRKs have only reported plant phenotypes related to development and/ or stress responses and have not investigated their downstream target proteins and finely tuned regulatory pathways. The generation of higher-order CRK mutants using the CRISPR system may overcome the possible functional redundancy of closely related CRKs. In addition, an inducible expression/knockdown system together with temporal sampling and data acquisition may overcome the pleiotropic effects of CRKs and reveal their direct molecular functions. Moreover, different CRK members, [Ca2+]cyt [45], apoplastic and/or cytoplasmic ROS bursts [22], and various phytohormone levels [87] form multiple complex feedback loops for positively or negatively modulating downstream gene expression and metabolism [99]. However, how CRKs respond to stress signals and regulate stress hormones to mediate energy and resource allocation during the stress growth balance requires further investigation [104]. Trends in Plant Science, Month 2023, Vol. xx, No. xx 15 Trends in Plant Science Elucidating CRK ligands and PTMs Some CRKs can form homodimers with themselves (e.g., CRK28 and CRK36) and/or heterodimers with closely related homologs (e.g., CRK28/CRK29, CRK39/CRK40, and CaCRK5/ CaCRK6), and interact with PRR components on the PM (e.g., CRK4, CRK6, CRK28, and CRK36) in response to pathogens and abiotic stress [2,11,38,39]. The formation of complexes enables rapid recruitment of signaling components to facilitate a robust defense response. Whether CRKs function as scaffolds in the PRR complex and/or sensors for recognizing extracellular ligands [39], and the specific ligands of the DUF26 domain that are crucial for CRK signaling, remain largely unknown [1,33]. Specific subcellular localizations [19], protein–protein interactions [39], and PTMs [38] of CRKs result in functional alterations and diverse physiological outputs. Thus, in-depth studies on the mechanisms of kinase activation, protein interactions, the dynamics of complex formation, and PTM crosstalk can greatly enhance our understanding of the key regulatory sites, conformational changes, and biological implications of different CRKs. Furthermore, single-cell-omic analysis, cellular and subcellular imaging, and proteomics-based CRK target mining in plants with different CRK genetic backgrounds may provide valuable insights into CRK signaling pathways and networks at the single-cell level [113]. Enhancing crop stress resilience through CRK regulation Given its vast genetic resources and fast life cycle, most of our knowledge about CRKs comes from studies in arabidopsis, especially regarding the roles of CRKs in immune responses. Whether this knowledge is translatable to crops is not known because the mechanisms of CRK functions might not be conserved in crops [11,33]. Therefore, testing the functions of CRKs in various crops upon disease and abiotic stress needs to be accomplished in due course. Ultimately, an improved understanding of CRK networks will facilitate synthetic biology and molecular design-based breeding toward improving crop stress resilience, yield, and quality. Acknowledgments We thank Miss Xianyao Huang and Miss Yuelu Zhao for figure preparation. This work was supported by the National Natural Science Foundation of China (grant 32070300), the Fund of Central Government Guides Local Science and Technology Development, China (YDZX20203100003927), the Fund of Shanghai Engineering Research Center of Plant Germplasm Resources, China (17DZ2252700) to S.D, the China Postdoctoral Science Foundation (2021M702202) to Y.Z., and the Faculty Start-Up Fund of the University of Mississippi to S.C. Declaration of interests The authors declare no conflicts of interest. 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