Catalytically inactive subgroup VIII receptor-like cytoplasmic kinases regulate the immune-triggered oxidative burst inArabidopsis thaliana

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Catalytically inactive subgroup VIII receptor-like cytoplasmic kinases regulate the immune-triggered oxidative burst in Arabidopsis thaliana View ORCID Profile Márcia Gonçalves Dias , View ORCID Profile Thakshila Dharmasena , Carmen Gonzalez-Ferrer , View ORCID Profile Jan Eric Maika , View ORCID Profile Maria Camila Rodriguez Gallo , View ORCID Profile Virginia Natali Miguel , View ORCID Profile Ruoqi Dou , View ORCID Profile Melissa Bredow , Kristen R Siegel , Richard Glen Uhrig , View ORCID Profile Rüdiger Simon , View ORCID Profile Jacqueline Monaghan doi: https://doi.org/10.1101/2024.05.30.596543 Márcia Gonçalves Dias 1 Department of Biology, Queen’s University , Kingston ON Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Márcia Gonçalves Dias Thakshila Dharmasena 1 Department of Biology, Queen’s University , Kingston ON Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thakshila Dharmasena Carmen Gonzalez-Ferrer 1 Department of Biology, Queen’s University , Kingston ON Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jan Eric Maika 2 Institute for Developmental Genetics, Heinrich Heine University , Düsseldorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jan Eric Maika Maria Camila Rodriguez Gallo 3 Department of Biological Sciences, University of Alberta , Edmonton, AB, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maria Camila Rodriguez Gallo Virginia Natali Miguel 1 Department of Biology, Queen’s University , Kingston ON Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Virginia Natali Miguel Ruoqi Dou 1 Department of Biology, Queen’s University , Kingston ON Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ruoqi Dou Melissa Bredow 1 Department of Biology, Queen’s University , Kingston ON Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Melissa Bredow Kristen R Siegel 1 Department of Biology, Queen’s University , Kingston ON Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Richard Glen Uhrig 3 Department of Biological Sciences, University of Alberta , Edmonton, AB, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rüdiger Simon 2 Institute for Developmental Genetics, Heinrich Heine University , Düsseldorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Rüdiger Simon Jacqueline Monaghan 1 Department of Biology, Queen’s University , Kingston ON Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jacqueline Monaghan For correspondence: jacqueline.monaghan{at}queensu.ca Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Protein kinases are key components of multiple cell signaling pathways. Several protein kinases of the receptor-like cytoplasmic kinase (RLCK) family have demonstrated roles in immune and developmental signaling across various plant species, making them a family of interest in the study of phosphorylation-based signal relay. Here, we present our investigation of a subfamily of RLCKs in Arabidopsis thaliana . Specifically, we focus on subgroup VIII RLCKs: MAZ and its paralog CARK6, as well as CARK7 and its paralog CARK9. We found that both MAZ and CARK7 associate with the calcium-dependent protein kinase CPK28 in planta, and furthermore that CPK28 phosphorylates both MAZ and CARK7 on multiple residues in areas that are known to be critical for protein kinase activation. Genetic analysis suggests redundant roles for MAZ and CARK6 as negative regulators of the immune-triggered oxidative burst. We find evidence that supports homo– and hetero-dimerization between CARK7 and MAZ, which may be a general feature of this protein family. Multiple biochemical experiments suggest that neither MAZ nor CARK7 demonstrate catalytic protein kinase activity in vitro. Interestingly, we find that a mutant variant of MAZ incapable of protein kinase activity is able to complement maz-1 mutants, suggesting noncatalytic roles of MAZ in planta . Overall, our study identifies subgroup VIII RLCKs as new players in Arabidopsis immune signaling and highlights the importance of noncatalytic functions of protein kinases. Introduction Phosphorylation is one of the most influential post-translational modifications shaping eukaryotic proteomes. Phosphorylated proteoforms can differ in their activation status, localization, stability, or ability to interact with other proteins, increasing the diversity of functions attributed to the products of single genes. Moreover, phosphorylation events occur in a cell-autonomous manner, which is critical to the ability of individual cells to respond to stress signals quickly. Phosphorylation is catalyzed by protein kinases that hydrolyze the γ-phosphate from ATP and transfer it to substrate proteins, typically on serine, threonine, or tyrosine residues. Compared to the metazoan protein kinase superfamily, the plant protein kinase superfamily is hugely expanded. For example, while there are ∼500 protein kinases encoded in the human genome, there are ∼1,400 protein kinases encoded in the genome of the angiosperm model plant Arabidopsis thaliana, and even more in plant genomes with higher ploidy levels. Within the superfamily, there are several subfamilies of protein kinases that differ in their enzymatic and substrate recognition properties, and can be phylogenetically organized based on their unique features ( Lehti-Shiu and Shiu, 2012 ). The largest protein kinase families in plants include transmembrane receptor-like kinases (RLKs) and cytoplasmic kinases of the mitogen-activated protein kinase (MAPK), Ca 2+ -dependent protein kinase (CDPK), and receptor-like cytoplasmic kinase (RLCK) subfamilies. A typical stress signaling pathway includes the activation of an RLK by binding an extracellular ligand followed by the trans-phosphorylation and activation of cytoplasmic protein kinases which then phosphorylate various substrates that result in signal transduction and temporary cellular reprogramming. As in many other stress pathways, protein kinases are integral to immune signaling. Immune sensing is achieved by pattern recognition receptors (PRRs) in the plasma membrane, many of which are either transmembrane RLKs or non-kinase receptor-like proteins (RLPs) ( DeFalco and Zipfel, 2021 ). Typically, serine/threonine protein kinases have an arginine preceding a conserved aspartate residue within their catalytic loops and are thus referred to as RD kinases ( Dardick and Ronald, 2006 ). Most immune-related PRRs are non-RD kinases that lack the aspartate residue and are less catalytically active than their RD counterparts ( Dardick and Ronald, 2006 ; Tang et al ., 2017 ). Due to this, PRRs generally function in large multiprotein immune complexes, and often require association with a co-receptor for signal transduction, such as BRASSINOSTEROID INSENSITIVE 1 ASSOCIATED KINASE 1 (BAK1) ( Nam and Li, 2002 ; Chinchilla et al ., 2007 ; Heese et al ., 2007 ; Monaghan and Zipfel, 2012 ). PRRs bind highly conserved microbial features known as microbe-associated molecular patterns (MAMPs) via their extracellular ligand-binding domains. Highly studied MAMPs include the 22 amino acid epitope of the bacterial motor protein flagellin (flg22), the 18 amino acid segment of elongation factor Tu (elf18), or components of bacterial or fungal cell walls ( Shu et al ., 2023 ). Following immune induction, endogenous small peptides called phytocytokines are released and can bind additional PRRs and amplify and propagate the signal ( Segonzac and Monaghan, 2019 ), together resulting in the activation of other protein kinases, the release of reactive oxygen species (ROS) into the apoplast, an increase in cytoplasmic Ca 2+ , and ultimately, transcriptional reprogramming ( DeFalco and Zipfel, 2021 ). Involved in growth, development and stress signaling, the 149 RLCKs in Arabidopsis cluster into 17 subgroups referred to as RLCK-II and RLCK-IV – RLCK-XIX ( Shiu and Bleecker, 2001 ; Liang and Zhou, 2018 ). The most studied RLCKs belong to the 46-member subgroup VII, including BOTRYTIS INDUCED KINASE 1 (BIK1) and closely related PBS1-like (PBL1) proteins ( Lu et al ., 2010 ; Zhang et al ., 2010 ), which form dynamic complexes with multiple PRRs ( Liang and Zhou, 2018 ). Signaling through BIK1 and PBL1 proteins is genetically required for immune responses, and these RLCKs are directly targeted by secreted pathogenic proteins known as effectors to suppress immune responses ( Veronese et al ., 2006 ; Lu et al ., 2010 ; Zhang et al ., 2010 ). Although BIK1 and related subgroup VII RLCKs have emerged as key regulators of immunity over the past decade, the earliest reports of RLCKs involved in pathogen defense surrounded the tomato kinases PSEUDOMONAS SYRINGAE PATHOVAR TOMATO (Pto) and FENTHION SENSITIVITY (Fen) ( Chandra et al ., 1996 ; Jia et al ., 1997 ). Both Pto and Fen interact with immune receptor PTO RESISTANCE AND FENTHION SENSITIVITY (Prf) ( Mucyn et al ., 2006 , 2009 ), which belongs to a cytoplasmic immune receptor family known as the nucleotide-binding, leucine-rich repeat (NLR) family. Whereas PRRs detect the presence of microbes in the extracellular space, NLRs detect the presence of pathogens that have actually infected cells ( Kourelis, 2023 ). Activated NLRs directly mount a robust immune response that results in cell death to clear the infection and protect neighbouring tissues. In this pathway, Pto and Fen are not classical ‘signal transducers’; rather, modification to their proteoforms serves as a marker for the presence of the secreted protein effectors AvrPto and AvrPtoB from the virulent bacterial pathogen Pseudomonas syringae pv. tomato that in turn activates Prf ( Mucyn et al ., 2006 , 2009 ). In addition, several RLCKs have been shown to function as decoy substrates for effectors that result in NLR activation ( Wang et al ., 2015 ; Martel et al ., 2020 ), illustrating the varied and complex roles that RLCKs play in immune signaling. BIK1 protein accumulation is controlled through an intricate interplay between phosphorylation and ubiquitination, which is thought to optimize immune signaling ( Dias et al ., 2022 ). Unactivated BIK1 is polyubiquitinated by related E3 ubiquitin ligases PLANT U-BOX 25 (PUB25) and PUB26 ( Wang et al ., 2018 a ). The calcium-dependent protein kinase CPK28 associates with and phosphorylates BIK1, PUB25, and PUB26 ( Monaghan et al ., 2014 ; Wang et al ., 2018 a ). Although we are still investigating the function of CPK28-mediated phosphorylation on BIK1, it has been shown that CPK28-mediated phosphorylation on PUB25 and PUB26 results in their activation and subsequent polyubiquitination of BIK1 ( Wang et al ., 2018 a ). We recently showed that CPK28 can also phosphorylate the Raf-like kinases MRK1, RAF26, and RAF39, which function in stomatal immunity ( Dias et al ., 2023 ). Here, we focus on the subgroup VIII RLCKs MAZZA (MAZ) and CYTOSOLIC ABA RECEPTOR KINASE 7 (CARK7) as novel substrates of CPK28. We uncover a role for MAZ and its paralog CARK6 as redundant negative regulators of the immune-triggered oxidative burst. Although they possess all hallmarks of active kinases, we find that MAZ and CARK7 do not possess detectable kinase activity in vitro, and that catalytic activity of MAZ is not required for its biological activity in vivo . Our work therefore highlights the importance of verifying catalytic activity prior to drawing conclusions about protein function and provides evidence for noncatalytic functions of protein kinases in signaling pathways. Results & Discussion CPK28 associates with MAZ and CARK7 in planta We recently identified five RLCKs as putative binding proteins of CPK28 in a proteomics screen following affinity purification of CPK28-YFP from cpk28-1/35S:CPK28-YFP transgenic lines ( Dias et al ., 2023 ). This included two members of RLCK subgroup VIII: MAZ and CARK7 ( Figure 1A-B ). Importantly, we did not identify MAZ or CARK7 peptides when we similarly affinity-purified two unrelated plasma membrane resident proteins – Lti6B-GFP ( Cutler et al ., 2000 ) from Col-0/ 35S:Lti6B-GFP and NSL1-YFP ( Holmes et al ., 2021 ) from nsl1-1/35S:NSL1-YFP ( Figure 1A ) ( Dias et al ., 2023 ), suggesting specificity. Subgroup VIII RLCKs have been studied in multiple species, including Arabidopsis, soybean, rice, and corn, with demonstrated roles in RLK-mediated signaling networks involved in immunity and development ( Liang and Zhou, 2018 ). For example, the tomato subgroup VIII RLCKs Pto-interacting protein 1 (SlPti1) and SlPti1b are required for the induction of immune-induced oxidative burst and resistance to infection with P. syringae pv. tomato ( Schwizer et al ., 2017 ). Moreover, SlPti1 associates with and is phosphorylated by SlPto ( Zhou et al ., 1995 ). Although originally named PTI1-like kinases in Arabidopsis (PTI1-1 – PTI1-11) ( Anthony et al ., 2006 ; Huangfu et al ., 2021 ), this subfamily was recently renamed CARKs ( Zhang et al ., 2018 ), and individual members of the subfamily have additional names. The eleven Arabidopsis subgroup VIII RLCKs cluster into three groups ( Figure 1B ) ( Herrmann et al ., 2006 ). The subgroup VIII-I protein MARIS/CARK4 regulates cell wall integrity sensing resulting in pollen tube and root hair defects ( Boisson-Dernier et al ., 2015 ; Liao et al ., 2016 ). The subgroup VIII-II RLCK CARK1 regulates abscisic acid (ABA) signaling through direct regulation of multiple ABA receptors ( Zhang et al ., 2018 ; Li et al ., 2019 , 2022 ). The subgroup VIII-III RLCK CARK8, and to a lesser extent CARK3, CARK7, and MAZ, may be involved in reactive oxygen stress signaling mediated by the kinase OXIDATIVE SIGNAL INDUCIBLE 1 (OXI1) ( Anthony et al ., 2006 ; Forzani et al ., 2011 ). In our previous work, we uncovered that the subgroup VIII-III protein MAZ is a component of stomatal and root development signaling mediated by CLAVATA1 (CLV1) family receptors that perceive CLV3/EMBRYO SURROUNDING REGION-RELATED (CLE) peptides ( Blümke et al ., 2021 ). Given the broad roles for subgroup VIII RLCKs in cellular signaling, we were motivated to study MAZ, CARK7, and related proteins in more detail. Download figure Open in new tab Figure 1. CPK28 associates with and phosphorylates MAZ and CARK7. (A) Number of unique peptides matching MAZ and CARK7, as well as peptides that match both MAZ and CARK7, identified by LC-MS/MS after affinity purification of CPK28-YFP, compared to Lti6B-GFP and NSL1-YFP as controls. R1, R2, and R3 refer to independent replicates. The full dataset has been previously published ( Dias et al ., 2023 ). (B) Phylogenetic tree of the Arabidopsis subgroup VIII RLCK family, with BIK1 as an outgroup. The tree was constructed using maximum likelihood with 1000 bootstraps in MEGAX; generated by JM. (C) FLIM-FRET analysis of proteins expressed in N. benthamiana . The fluorescence lifetime (ns) of MAZ-GFP and CARK7-GFP is reduced only in the presence of CPK28-mCherry and not when expressed alone or in the presence of Lti6B-mCherry. Representative confocal micrographs are displayed above the plot. At least 20 independent cells were measured for each; statistically significant groups are indicated by lower-case letters based on a one-way ANOVA and Tukey’s post-hoc test ( p <0.002). Data collected by JEM. (D-E) In vitro kinase assays using CPK28 as the kinase and MAZ K141N (D) or CARK7 K97N (E) as substrates, visualized using Phostag gel staining. Coomassie Brilliant Blue (CBB) indicates loading. These experiments were repeated more than 4 times by TD over the course of a year and representative blots are shown. To confirm that CPK28 is able to associate with MAZ and CARK7, we first employed split-luciferase complementation analysis (SLCA) using transient expression in Nicotiana benthamiana . Here, the N-terminal canonical domain of firefly luciferase (nLuc) is translationally fused to the C-terminus of one protein of interest, while the C-terminal canonical domain of luciferase (cLuc) is N-terminally fused to another protein of interest. If the two proteins associate, a functional luciferase is formed that can produce light if given the substrate luciferin. For these experiments, we used the RLK FERONIA (FER) as a control because it is similarly located at the plasma membrane. We found that co-expressing CPK28-nLuc and cLuc-MAZ reconstituted luciferase function, whereas co-expressing FER-nLuc and cLuc-MAZ did not ( Supplemental Figure S1A ). Similarly, luciferase was reconstituted when we co-expressed CPK28-nLuc and cLuc-CARK7, but not when we co-expressed FER-nLuc and cLuc-CARK7 ( Supplemental Figure S1B ). We next tested if CPK28 can associate with MAZ and CARK7 using fluorescence lifetime imaging-based Förster resonance energy transfer (FLIM-FRET) measurements. In FRET experiments, two proteins of interest are tagged with distinct fluorescent proteins that have overlapping excitation and emission spectra. The phenomenon occurs as a radiation-free energy transfer from a ‘donor’ fluorophore to an ‘acceptor’ fluorophore if they are in close proximity and appropriate orientation. FLIM measures the decrease in the excited-state lifetime of the donor fluorophore when in the presence of an acceptor and can provide information about protein-protein interactions ( Spatola Rossi et al ., 2022 ). Here, we transiently co-expressed CPK28-mCherry or Lti6B-mCherry with either MAZ-GFP or CARK7-GFP in N. benthamiana . We found that the fluorescence lifetime of both MAZ-GFP and CARK7-GFP was reduced in the presence of CPK28-mCherry but not Lti6B-mCherry ( Figure 1C ), suggesting that CPK28 is sufficiently close to MAZ and CARK7 to allow energy transfer. Moreover, confocal micrographs suggest that this association occurs in the plasma membrane, which is where CPK28 and MAZ localize ( Monaghan et al ., 2014 ; Blümke et al ., 2021 ). We conclude that CPK28 is able to associate with both MAZ and CARK7 in planta . CPK28 phosphorylates MAZ and CARK7 on multiple residues in vitro Several in vivo and in vitro phosphorylation sites have been mapped on subgroup VIII RLCKs from various phosphoproteomics screens ( Supplemental Figure S2 ), suggesting that they may be regulated by phosphorylation. CPK28 is an active protein kinase ( Matschi et al ., 2013 ; Monaghan et al ., 2014 ) that phosphorylates several target proteins ( Wang et al ., 2018 a ; Ding et al ., 2022 ; Dias et al ., 2023 ) including the subgroup VII RLCK BIK1 ( Monaghan et al ., 2014 ), which is closely related to MAZ and CARK7. Because they associate, we reasoned that CPK28, MAZ, and CARK7 may engage in trans-phosphorylation. We first tested if CPK28 can phosphorylate MAZ and CARK7. As MAZ and CARK7 are protein kinases, we generated catalytically inactive variants His 6 -MAZ K141N and His 6 -CARK7 K97N (in which the ATP-binding Lys has been mutated to Asn) to use as substrates for CPK28. We found that CPK28 was able to phosphorylate both MAZ K141N and CARK7 K97N in an ATP-dependent manner, as visualized by Phostag staining ( Figure 1D-E ). To test if MAZ and CARK7 are able to phosphorylate CPK28, we used the catalytically inactive His 6 -CPK28 D188A variant (in which a critical Asp in the activation loop has been mutated to Ala) ( Matschi et al ., 2013 ; Monaghan et al ., 2014 ) as substrate. We found that neither MAZ nor CARK7 were able to reciprocally phosphorylate CPK28 D188A ( Supplemental Figure S1C-D ). To identify which residues are phosphorylated by CPK28, we performed additional kinase assays using MAZ K141N and CARK7 K97N as substrates and analyzed phosphopeptides by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). We identified nine phosphosites on MAZ and seven phosphosites on CARK7, four of which were in conserved positions on MAZ and CARK7 ( Figure 2A-B ). Interestingly, the phosphosites were located in regions that are considered critical for kinase activation, including in the glycine-rich loop, active site, and in the activation loop ( Figure 2B ). We conclude that CPK28 is able to unidirectionally phosphorylate MAZ and CARK7 on multiple residues. Download figure Open in new tab Figure 2. CPK28 phosphorylates MAZ and CARK7 on multiple residues. (A) Unique MAZ and CARK7 phosphopeptides following kinase assays with CPK28. Each peptide was present in at least 2/3 independent replicates and not found in control samples with ATP but without CPK28. The bolded sites are the phosphosites and their positions are indicated on the right. Kinase assays performed by TD; trypsin digests and LC-MS/MS performed by MCRG. (B) Sequence alignment between MAZ and CARK7 with protein secondary structures indicated as visualized by ESPript ( Robert and Gouet, 2014 ). Residues phosphorylated by CPK28 are indicated in pink (CARK7 sites on top and MAZ sites on the bottom). Analysis by JM. MAZ and CARK6 inhibit immune-triggered ROS Given the documented roles of RLCKs in immune signal transduction ( Liang and Zhou, 2018 ), we sought to assess if subgroup VIII RLCKs play a role in immune signaling. We obtained two insertional mutants in each of PTI1-3/CARK5/MAZZA and its paralog PTI1-5/CARK6, as well as PTI1-1/CARK7 and its paralogs PTI1-2/CARK8 and PTI1-6/CARK9 ( Figure 3A ) . While we were unable to identify any lines carrying the annotated T-DNA insertion in cark8-1 (WDL_Hs008-06A) , cark8-2 (Sail_147C05), or cark9-2 (Salk_079894), we were able to identify homozygous alleles in all other lines and confirmed that their target genes were downregulated ( Figure 3B-E ). Expecting some genetic redundancy between paralogs, we also generated maz-1 cark6-2 and cark7-1 cark9-1 double mutants. Leaf morphology for all single and double mutant lines was indistinguishable from Col-0 wild-type ( Figure 3F ). Download figure Open in new tab Figure 3. MAZ and CARK6 inhibit immune-triggered ROS. (A) Schematic representation, drawn to scale, of Arabidopsis subfamily VIII RLCK genes, indicating exons (boxes), untranslated regions (lines), and the location of T-DNA insertion alleles. Genomic information was retrieved from The Arabidopsis Information Resource (TAIR) by CGF and JM. Lines were genotyped to homozygosity by CGF as outlined in Supplemental Table S1 . (B-E) Quantitative real-time qRT-PCR of MAZ (B) , CARK6 (C) , CARK7 (D) , and CARK9 (E) , relative to UBOX. Means for 3 independent biological replicates are shown +/-standard error of the mean. All data collected by CGF. Primers for genotyping and qRT-PCR are provided in Supplemental Table S1 . (F) Photographs of representative plants of each genotype after 5 weeks of growth on soil under short-day conditions. Photographs taken by MGD. (G) ROS production measured in relative light units (RLUs) after treatment with 100 nM flg22. Values represent means +/-standard error (n=6 leaf discs). Assays were repeated several times by MGD and JM over multiple years; representative data is shown. (H) Growth of Pseudomonas syringae pv. tomato ( P.s.t. ) isolate DC3000 3 days after syringe-inoculation. Data from 3 independent biological replicates are plotted together, denoted by black, teal, and magenta dots. Values are colony forming units (cfu) per leaf area (cm 2 ) from 4-5 samples per genotype (each sample contains 3 leaf discs from 3 different infected plants). The line represents the mean (n=12). Lower case letters indicate that all genotypes are in the same statistical group, as determined by a one-way ANOVA followed by Tukey’s post-hoc test. Data collected by MGD. A burest of reactive oxygen species (ROS) is readily observable within the first 5-30 min following exposure to immunogenic molecules such as bacterial flagellin ( Gómez-Gómez et al ., 1999 ). We did not observe any differences in flg22-induced ROS in the maz-1, maz-2, cark6-1, cark6-2, cark7-1, cark7-2, or cark9-1 single mutants, nor in the cark7-1 cark9-1 double mutant, compared to Col-0 ( Figure 3G ; Supplemental Figure S3A-E ). We did, however, observe enhanced flg22-induced ROS in the maz-1 cark6-2 double mutant ( Figure 3G ; Supplemental Figure S3E ). The rapid phosphorylation and activation of MAPKs occurs in parallel with the oxidative burst, also observable within the first 5-30 minutes of exposure to immunogenic peptides ( Asai et al ., 2002 ; Son et al ., 2011 ). We found that the activation of MPK3/6 and MPK4 was similar in Col-0, maz-1 cark6-2 , and cark7-1 cark9-1 double mutants (Supplemental Figure S3F) , suggesting that subgroup VIII RLCKs do not function in the parallel MAPK pathway. As both ROS and MAPK activation are considered ‘early’ immune responses ( Yu et al ., 2017 ), we extended our study to also include ‘later’ responses, including both immune-triggered inhibition of seedling growth and resistance to a bacterial pathogen. In Col-0 seedlings, continual exposure to 100 nM flg22 for 10-14 days results in 60-90% reduction in seedling weight ( Supplemental Figure S3G ) ( Gómez-Gómez et al ., 1999 ). This effect was similar in both maz-1 cark6-2 and cark7-1 cark9-1 double mutants ( Supplemental Figure S3G ). In addition, we did not observe any differences between Col-0 and the double mutants when we infected adult plants with the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 ( Figure 3H ). Together, these data suggest that MAZ and CARK6 inhibit the immune-triggered oxidative burst but that this does not correlate to enhanced resistance against P.s.t. DC3000. Interestingly, while the subgroup VIII RLCKs SlPti1a and SlPTi1b from tomato ( Zhou et al ., 1995 ; Schwizer et al ., 2017 ; Giska and Martin, 2019 ), NbPti1b and NbPti1c from N. benthamiana ( Schwizer et al ., 2017 ), and CsPti1-L from cucumber ( Oh et al ., 2014 ) promote immune signaling, the subgroup VIII RLCK OsPti1a negatively regulates immunity in rice ( Takahashi et al ., 2007 ; Matsui et al ., 2010 ). Overexpression of OsPti1a reduces resistance to rice blast fungus, while OsPti1a knockouts are characterized by chlorotic lesions and other hallmarks of deregulated immune signaling ( Takahashi et al ., 2007 ). The hyperimmunity and enhanced resistance observed in Ospti1a knockouts can be suppressed by silencing OsRAR1 ( REQUIRED FOR AVRB-DEPENDENT SUSCEPTIBILITY AND CHLOROSIS ), a well-known downstream component of NLR signaling ( Shirasu, 2009 ). Therefore it is also possible that OsPti1a is guarded by an NLR ( Takahashi et al ., 2007 ; Schwizer et al ., 2017 ) that is present in rice but absent in solanaceous plants ( Schwizer et al ., 2017 ). Our finding that MAZ and CARK6 negatively regulate immune triggered ROS suggests roles for subgroup VIII as negative regulators of defense. Although we were unable to obtain cark8 mutants, previous work has shown that CARK8 activity increases in response to stress signals including phosphatidic acid, hydrogen peroxide, and the bacterial immunogenetic elicitors xylanase and flagellin ( Anthony et al ., 2006 ), which may suggest a role in immune signaling. As this subfamily is large in Arabidopsis, and as genetic redundancy is well-known amongst other RLCKs ( Rao et al ., 2018 ), it is likely that a quintuple mutant lacking MAZ, CARK6, CARK7, CARK8, and CARK9 may reveal stronger immune phenotypes. We propose this as an avenue for future analysis of this gene family. MAZ and CARK7 are catalytically inactive in vitro To assess if MAZ and CARK7 possess catalytic autophosphorylation activity, we performed in vitro kinase assays using recombinant His 6 -MAZ and His 6 -CARK7. We included either GST-BIK1 or His 6 -CPK28 as technical controls for our assays. Although we could readily detect autophosphorylation on BIK1, we were unable to detect autophosphorylation on MAZ or CARK7 when we supplied the kinases with divalent magnesium or manganese as cofactors along with [γ32P]-ATP ( Figure 4A ). Because some protein kinases are inhibited by phosphorylation, we next expressed and purified the recombinant proteins from an E. coli strain that expresses λ-phosphatase. However, we could again not detect any in vitro autophosphorylation for both MAZ or CARK7 ( Figure 4B ). Some protein kinases do not autophosphorylate but can transphosphorylate substrates. We therefore assessed if MAZ or CARK7 are able to trans-phosphorylate Histone 3S (a highly phosphorylatable protein considered a universal substrate for protein kinases), however we once again could not detect any activity ( Figure 4C ). We note that a previous study reported that both MAZ and CARK7 (as well as CARK2, CARK4, and CARK11) are able to phosphorylate multiple ABA receptors ( Li et al ., 2022 ), however in our view these assays were not adequately controlled and are hard to interpret. The CARKs and ABA receptors were combined in the presence or absence of ATP and phosphorylation was visualized using an anti-pThr antibody. Immunoreactive bands are clearly visible for the ABA receptors only in the presence of both CARKs and ATP, however there is no control with the receptors in the presence of ATP and absence of CARKs. We find it difficult to reconcile differences between our studies. We also note that another study showed that CARK7, CARK8, and MAZ exhibit low levels of autophosphorylation ( Anthony et al ., 2006 ). However, those experiments were performed following transfection of Arabidopsis protoplasts, which may not faithfully reflect autophosphorylation. In the same study, the authors showed that CARK7 and CARK8 can be phosphorylated by OXI1 ( Anthony et al ., 2006 ), a protein kinase involved in signaling events broadly mediated by reactive oxygen species ( Rentel et al ., 2004 ). Download figure Open in new tab Figure 4. MAZ and CARK7 are catalytically inactive. (A-B) In vitro autophosphorylation assays using His 6 -MAZ and His 6 -CARK7 alongside GST-BIK1 as a control. Experiments in (A) compare the utility of Mn 2+ or Mg 2+ as co-factors, while experiments in (B) compare proteins purified from E. coli BL21 (may already be phosphorylated) with those purified from an E.coli BL21 strain expressing λ-phosphatase (to dephosphorylate proteins). Autoradiographs (autorad) indicate incorporation of γP32 and protein loading is indicated by post-staining the membranes with Coomassie Brilliant Blue (CBB). These assays were repeated more than three times by TD; representative experiments are shown. (C) In vitro transphosphorylation assays between His 6 -MAZ and His 6 -CARK7 and the universal substrate H3S, alongside transphosphorylation of H3S by His 6 -CPK28. Autorad indicates incorporation of γP32 and protein loading is indicated with CBB. These assays were repeated more than three times by TD; representative experiments are shown. (D) FLIM-FRET analysis of proteins expressed in N. benthamiana . The fluorescence lifetime (ns) of MAZ-GFP and CARK7-GFP is reduced only in the presence of CARK7-mCherry and not when expressed in the presence of Lti6B-mCherry. Representative confocal micrographs are displayed above the plot. At least 20 independent cells were measured for each; statistically significant groups are indicated by lower-case letters based on a one-way ANOVA and Tukey’s post-hoc test (p<0.0001). Data collected by JEM. (E) In vitro transphosphorylation assays between His 6 -MAZ and His 6 -CARK7. Autorad indicates incorporation of γP32 and protein loading is indicated with CBB. These assays were repeated more than three times by TD; representative experiments are shown. (F) Protein structure overlays comparing the AlphaFold-predicted structures of CARK7 and MAZ with the solved structure of CARK1 (PBD: 5XD6), determined using the matchmaker command in ChimeraX ( Pettersen et al ., 2021 ). The root-mean-square deviation (RMSD) of <0.7 Å indicates a high level of similarity. (G-H) In vitro autophosphorylation assays using His 6 -MAZ Δ90 (G) and His 6 -MAZ N256G (H) alongside GST-BIK1 as a control. Autorad indicates incorporation of γP32 and protein loading is indicated with CBB. Data in (G) was collected by TD using protein purified by VNM, while data in (H) was collected by TD using protein purified by TD. Assays were repeated more than three times and representative experiments are shown. (I-J) In vitro trans-/auto-phosphorylation assays between His 6 -CPK28 and His 6 -MAZ or His 6 -MAZ K141N (I) , or His 6 -CARK7 or His 6 -CARK7 K97N (J). Autorad indicates incorporation of γP32 and protein loading is indicated with CBB. These assays were repeated more than three times by TD; representative experiments are shown. Previous work has shown that many CARKs are able to associate with themselves and each other ( Li et al ., 2022 ), suggesting that homo– and/or hetero-dimerization may be an important feature of subgroup VIII RLCKs. For example, MAZ can associate with itself ( Blümke et al ., 2021 ) as well as CARK6 ( Li et al ., 2022 ), and CARK7 can associate with CARK11 ( Li et al ., 2022 ). To test if MAZ can associate with CARK7, we again employed SLCA and found that co-expression of CARK7-nLuc and cLuc-MAZ successfully reconstituted luciferase function, whereas co-expression of FER-nLuc and cLuc-MAZ did not ( Supplemental Figure S4A ). Similarly, in FLIM-FRET experiments, we found that the lifetime of MAZ-GFP was reduced when co-expressed with CARK7-mCherry, but not when co-expressed with Lti6B-mCherry ( Figure 4D ). In addition, we observed CARK7 self-association in both SLCA ( Supplemental Figure S4B ) and FLIM-FRET ( Figure 4D ) experiments. We conclude that CARK7 and MAZ are able to associate with themselves and each other, possibly forming homo– and hetero-dimers, which may reflect a general feature of this protein family. To test the hypothesis that heterodimer formation may regulate MAZ and CARK7 catalytic activity, we next conducted kinase assays with both proteins together. However, we were again unable to detect phosphoryl transfer ( Figure 4E ). Overall, we conclude that MAZ and CARK7 are catalytically inactive in vitro . These results were unexpected since most studied RLCKs possess kinase activity ( Liang and Zhou, 2018 ). We therefore interrogated the sequences of subgroup VIII RLCKs in detail in an attempt to uncover why they are unable to catalyze phosphorylation in vitro. A survey of the literature revealed that while several subgroup VIII RLCKs from multiple species do autophosphorylate in vitro ( Zhou et al ., 1995 ; Anthony et al ., 2006 ; Herrmann et al ., 2006 ; Zou et al ., 2006 ; Takahashi et al ., 2007 ; Forzani et al ., 2011 ; Oh et al ., 2014 ; Zhang et al ., 2018 ; Giska and Martin, 2019 ; Li et al ., 2019 ; Peng et al ., 2022 ), others do not ( Staswick, 2000 ; Tian et al ., 2004 ; Liao et al ., 2016 ). We therefore decided to take a comparative approach. The protein structures predicted by AlphaFold ( Jumper et al ., 2021 ) for MAZ and CARK7 indicate that both are likely to fold into canonical protein kinases with well-structured N– and C-lobes. We superimposed the predicted structures for MAZ and CARK7 with the experimentally-determined structure for CARK1 (PBD: 5XD6) which has autophosphorylation activity ( Zhang et al ., 2018 ), and found a high level of similarity, with minimal root mean square deviations less than 0.7 Å ( Figure 4F ). The only differences we observed were in intrinsically disordered or low-order regions such as the activation loops which have low confidence prediction values and were missing in the CARK1 structure ( Zhang et al ., 2018 ). Based on this comparison, we surmise that MAZ and CARK7 are likely to fold into proteins that are structurally similar to CARK1. In this regard, it is interesting to note that homo– and hetero-dimerization of CARK1 and CARK3, as well as the association between CARK1 and ABA receptors, is dependent on the presence of catalytic residues ( Zhang et al ., 2018 ; Li et al ., 2019 , 2022 ). This may suggest that at least some subgroup VIII RLCKs adopt protein conformations that are sensitive to changes in the catalytic cleft. Like many RLCKs, MAZ and CARK7 contain a central protein kinase domain flanked by variable N– and C-terminal domains. In the specific case of MAZ and CARK7, the C-terminal domain is very short, but the N-terminal domains are longer (∼100 in MAZ ( Blümke et al ., 2021 ) and ∼50 aa in CARK7) and are predicted to be intrinsically disordered. We hypothesized that the N-terminal extensions may inhibit kinase activity in some way. Using MAZ as a case study, we generated a mutant variant translationally fused to His 6 but lacking the majority of the N-terminal domain (starting at L90 following M1; 10 amino acids N-terminal to the canonical kinase domain). We were again unable to detect any autophosphorylation on MAZ Δ90 ( Figure 4G ), arguing against an N-terminal auto-inhibition mechanism. We next generated a multiple sequence alignment of the entire subgroup VIII RLCK subfamily from plants spanning the plant kingdom: dicotyledonous angiosperms Amborella trichopoda (3 members), Arabidopsis thaliana (11 members), Solanum lycopersicum (7 members), Glycine max (11 members), and Cucumis sativus (3 members); monocotyledonous angiosperms Oryza sativa (10 members) and Zea mays (13 members); moss Physcomitrium patens (2 members), and liverwort Marchantia polymorpha (1 member). In all cases, we could identify key features of active protein kinases ( Nolen et al ., 2004 ): in the N-lobe, we identified the glycine-rich loop and the ATP-binding lysine; in the C-lobe, we identified the ‘HRD’ motif and the critical Asp residue of the ‘DFG’ motif in the activation loop. We noted, however, that all subgroup VIII RLCKs do not possess typical DFG motifs, as the Gly is either replaced by Asn or Asp, resulting in non-canonical DFN or DFD motifs that correspond to phylogenetic clusters within the subgroup VIII RLCK family ( Supplemental Figure S5 ). Subgroup VIII-III RLCKs, including MAZ and CARK7, contain DFN. For many protein kinases, the precise positioning of the DFG-Phe is critical for enzyme activation as it subsequently allows for the correct positioning of the DFG-Asp to bind ATP in the active site ( Ung and Schlessinger, 2015 ). Both Asn and Asp are larger amino acids than Gly, which may result in steric hindrance of the neighbouring Phe in the DFN and DFD variants. We therefore generated a variant of MAZ with a canonical DFG motif, His 6 -MAZ N256G , and assessed autophosphorylation in vitro. However, we were once again unable to detect any activity ( Figure 4H ). Because CPK28 phosphorylates MAZ and CARK7 on residues known to be critical for kinase activation ( Figure 2 ), we hypothesized that they may require transphosphorylation for activation. We therefore performed additional kinase assays using MAZ or CARK7 as substrates for CPK28 in comparison to MAZ K141N or CARK7 K97N . We reasoned that if MAZ or CARK7 required phosphorylation by CPK28 for activation, we should observe an increase in the incorporation of [γ32P]-ATP on the wildtype variants compared to the catalytically inactive variants, reflecting both transphosphorylation events by CPK28 as well as autophosphorylation events by MAZ or CARK7. However, we did not observe any noticeable difference between the substrates ( Figure 4I-J ). This suggests that phosphorylation by CPK28 is not sufficient to activate the enzymatic function of MAZ or CARK7. Overall, after testing several hypotheses, we conclude that neither MAZ nor CARK7 display catalytic activity in vitro . The catalytic activity of MAZ is not required for its role in the CLV3p signaling pathway Although we could not detect any activity in vitro, it is possible that MAZ, CARK7, and other subgroup VIII RLCKs are active kinases in vivo under specific contexts. For example, they may require cellular cues or activation by upstream protein kinases or binding partners similar to mitogen-activated or cyclin-dependent protein kinases (MAPKs and CDKs). If this is the case, it is reasonable to expect their catalytic activity to be essential to carry out their biological roles. Although our analysis above indicates redundant roles between MAZ and CARK6 in immune-triggered ROS ( Figure 3G ; Supplemental Figure S3E ), we recently showed that maz-1 single mutants are partially resistant to CLV3p-triggered root meristem differentiation, which is genetically complemented in both maz-1/pUBQ10:MAZ-GFP and maz-1/pMAZ:MAZ-mCherry transgenic lines ( Blümke et al ., 2021 ). To test if kinase activity is required for the biological function of MAZ, we generated two independent and homozygous maz-1/p35S:MAZ K141N -GFP transgenic lines. Both lines grew similarly to Col-0 with no aberrant leaf morphology or any obvious growth defects ( Figure 5A ), and we verified that the transgenic lines express MAZ K141N -GFP at the expected size of ∼74 kDa ( Figure 5B ) . Interestingly, in both cases, expression of p35S:MAZ K141N -GFP was able to fully restore sensitivity to CLV3p in maz-1 in a manner comparable to pUBQ:MAZ-GFP and pMAZ:MAZ-mCherry ( Figure 5C ). These results suggest that the catalytic activity of MAZ is dispensable for its role in CLV1f-mediated signaling. As we were unable to assess if catalytic activity of MAZ is required for its role in immune-triggered ROS using these lines, it is unclear if MAZ and/or CARK6 require catalytic activity in immune signaling. Nevertheless, these results demonstrate that the biological function of MAZ involves a noncatalytic mechanism. Download figure Open in new tab Figure 5. The catalytic activity of MAZ is not required for its role in the CLV3p signaling pathway. (A) Photographs of representative plants of each genotype after 5 weeks of growth on soil under short-day conditions. Transgenic lines were generated and photographed by MGD. (B) MAZ K141N -GFP (∼74 kDa) migrated to its expected size in the maz-1/35S:MAZ K141N -GFP transgenic lines. Coomassie Brilliant Blue (CBB) of RuBisCO indicates loading. This experiment was repeated twice by MGD with identical results. (C) Root length of 55-119 10-day-old seedlings grown on 0.5x MS agar plates supplemented with 10 nM CLV3p, relative to mean root length of 10-day-old seedlings grown on 0.5x MS agar with no peptide. Data from 3 independent biological replicates are plotted together, denoted by black, teal, and magenta dots. Lower case letters indicate statistically significant groups, determined by a one-way ANOVA followed by Tukey’s post-hoc test (p<0.0001). Data collected by MGD. Noncatalytic roles for protein kinases are now well recognized, including roles as allosteric regulators and protein scaffolds ( Kung and Jura, 2016 ). Recently, the LRR-RLK EFR (ELONGATION FACTOR TU RECEPTOR) was shown to undergo phosphorylation-mediated conformational changes that allow for the allosteric activation of its co-receptor BAK1 ( Bender et al ., 2021 ; Mühlenbeck et al ., 2024 ). Although EFR is an active kinase in vitro , mutant variants that impair catalytic activity in vivo remain capable of initiating immune signaling ( Bender et al ., 2021 ). The current model suggests that phosphorylation within the activation loop stabilizes an active conformation of EFR that allows it to allosterically activate BAK1 to initiate immune signaling ( Mühlenbeck et al ., 2024 ). Although we do not have any evidence to support a similar model for subgroup VIII RLCKs, it is possible that phosphorylation by upstream kinases such as CPK28 could help stabilize conformations of MAZ and CARK7 that are capable of allosterically regulating downstream partners, possibly also acting as scaffolds to enable or coordinate molecular complex formation. Methods Germplasm and plant growth conditions A. thaliana and N. benthamiana plants were grown in the Queen’s Phytotron Facility or in phytochambers at Heinrich Heine University. For aseptic growth, A. thaliana seeds were surface sterilized using a 40% bleach solution and sown onto petri plates with 0.5x Murashige and Skoog (MS) media containing 0.8% agar. Afterward, they were subjected to cold stratification in darkness at 4°C for 3-5 days before being exposed to light. For plants grown in soil, seeds were directly sown onto potting soil (Sungro Sunshine Mix 1 or Fafard’s Agro G6 with Coco). Seedlings were later transplanted either individually into 3” pots or six plants per 8” pot two weeks after sowing. Controlled growth chambers (BioChambers and Conviron) were utilized for plant cultivation, maintaining a 10-hour light and 8-hour dark cycle at 22°C, with 30% relative humidity and a light intensity of 150 µE m 2 s -1 . Plants were watered from the top as needed, typically every other day, and fertilized every two weeks with a solution containing 1.5 g/L of 20:20:20 N:P:K. N. benthamiana seeds followed a similar procedure, sown on potting soil and transplanted individually, but were cultivated in a dedicated growth chamber (Conviron) with a 16-hour light and 8-hour dark cycle, receiving weekly fertilization. Additionally, mite bags containing Amblyseius swirskii (Koppert) were introduced bi-weekly to each plant tray to prevent pest infestations. Information on all germplasm used in this study is outlined in detail in Supplemental Table S1 . Segregating T-DNA insertion lines were obtained from the Arabidopsis Biological Resource Centre (ABRC) and genotyped using gene-specific primers in standard polymerase chain reactions (PCR). To determine gene expression, we extracted RNA using Aurum Total RNA Mini Kit (BioRad), according to the manufacturer’s instructions. Reverse transcription (RT) was achieved using Superscript III (Invitrogen), according to the manufacturer’s instructions. Quantitative RT-PCR was performed using Sso Advanced Universal SYBR Green Supermix (BioRad) according to the manufacturer’s directions, on a CFX96 Touch Real-Time PCR Detection System (BioRad), using primers specific to each gene as outlined in Supplemental Table S1 . Higher-order mutants were generated by crossing and genotyping to homozygosity in the F 2 and F 3 generations. Transgenic plants were generated by floral dip with Agrobacterium tumefaciens GV3101 carrying a suitable binary vector for selection, as previously described ( Clough and Bent, 1998 ). Independent lines were followed through four generations and genotyped to homozygosity based on 100% resistance to the selection marker in the T 3 generation. All details relating to the CPK28 proteomics screen are described in full elsewhere ( Dias et al ., 2023 ). Molecular cloning Detailed information about all vectors used in this study, including previously published vectors, can be found in Supplemental Table S1. Recombination of entry vectors into Gateway-compatible binary vectors pK7FWG2 ( Karimi et al ., 2002 ), pABindGFP ( Bleckmann et al ., 2010 ), or pABindmCherry ( Bleckmann et al ., 2010 ) was achieved using Gateway LR Clonase II (Invitrogen) according to the manufacturer’s instructions. Directional cloning by digestion-ligation into pCAMBIA1300-nLuc and pCAMBIA1300-cLuc ( Chen et al ., 2008 ) was achieved using PCR amplification with Q5 Taq polymerase, construct-specific endonucleases and T4 DNA ligase (all enzymes from NEB BioLabs), according to the manufacturer’s directions. Successful assembly of all plasmids was verified using Sanger sequencing (Centre for Applied Genomics, Toronto ON, Canada) or whole-plasmid sequencing (Plasmidsaurus, Eugene OR, USA). For recombinant protein production in Escherichia coli , gene coding regions were synthesized and cloned into pET28a+ (Novagen) by Twist BioSciences (San Francisco, USA). Split-luciferase complementation A. tumefaciens GV3101 carrying plasmids for split-luciferase complementation was infiltrated into fully expanded leaves of 3-4 week old N. benthamiana plants alongside the viral suppressor P19 ( Voinnet et al ., 2003 ). Leaf discs were harvested after 3 days and exposed to 1 mM luciferin (Gold Biotechnology) for 10 minutes in the dark, followed by recording light emission (integration time of 1 sec) using the LUM module in a SpectraMax Paradigm Multi Mode Microplate Reader. FLIM-FRET Fluorescence lifetime was measured on a Zeiss LSM 780 confocal microscope (40× water immersion objective, Zeiss C-PlanApo, NA 1.2). For TCSPC, a PicoQuant Hydra Harp 400 (PicoQuant, Berlin, Germany) was used. Photon counting was performed with picosecond resolution. GFP was excited with a 485 nm pulsed polarized laser (LDH-D-C-485, 32 MHz, PicoQuant, Berlin, Germany). mCherry was excited with a 561 nm laser with 1% laser power. Laser power at the objective lens was adjusted to 1 µW for the 485 nm laser. Light, emitted from the sample, was separated by a polarizing beam splitter before photons were selected with a band-pass filter. For GFP, a 520/30 band-pass filter; and for mCherry, a 607/70 band-pass filter was used. When GFP served as a donor, a LP610 beam splitter was used. Photons were detected with Tau-SPADs (PicoQuant, Berlin, Germany). Images were acquired at zoom 8 resolution of 256 x 256 pixels with a pixel size of 0.1 µm and a pixel dwell time of 12.54 µs and laser repetition rate of 32 MHz. Photons were collected over 40 frames. To avoid pileup effects, nuclei containing high donor concentrations were avoided. Before image acquisition, the system was calibrated. For this, the objective was adjusted to reach a maximal count rate. FCS curves of Rhodamine110 dye and water were acquired to monitor the system function. Internal response functions for each laser were determined by measuring the fluorescence decay of quenched erythrosine in saturated KI using the same hardware settings as for the FRET pair. The fluorescence decays of selected ROIs in the FLIM image were analyzed with the SymPhoTime FLIM analysis software (SymPhoTime 64, version 2.4; PicoQuant, Berlin, Germany). TCSPC bins of channel 1 and 2 (parallel and perpendicular light) were binned by eight resulting in a bin width of 8 ps. Nuclei were selected by hand using the ROI tool. Chloroplasts and pixels above the pile-up limit (10% of the laser repetition rate) were manually removed. Decays from donor only samples were fitted with the FLIM analysis tool (Fitting model: n-exponential reconvolution). Judged by fitting residuals and Chi-square-test, one lifetime (model parameter n=1) was needed to fit donor only decays containing GFP. FRET samples (containing GFP and mCherry) as well as donor only samples (only GFP) were fitted using the LT FRET image analysis tool. Parameter “τ Donor ” was fixed as the average donor only lifetime measured beforehand in the FLIM analysis. The second lifetime parameter “τ FRET ” corresponds to the FRET fraction of the sample and was fitted within limits corresponding to 10% and 80% of the average lifetime acquired in the donor only samples. The lower limit of the amplitude of the FRET fraction “ A FRET ” was set to 0.0001 to avoid fitting of negative amplitudes in donor only samples and samples containing Lti6B-mCherry. Average amplitude weighted lifetimes were calculated as the sum of each lifetime component (τ FRET and τ donor ) weighted by their respective amplitude: Immune and growth assays Immune-triggered ROS, seedling growth inhibition, MAPK activation, and infection with Pseudomonas syringae pv. tomato DC3000 were performed as described previously ( Monaghan et al ., 2014 ; Bredow et al ., 2019 ; Dias et al ., 2023 ). CLV3p-induced root inhibition was performed as described previously ( Blümke et al ., 2021 ). The 22 amino acid flg22 peptide ( Gómez-Gómez et al ., 1999 ) was synthesized by EZ Biotech (Indiana USA), and the 13 amino acid CLV3p peptide ( Kondo et al ., 2006 ) was synthesized by LifeTein (New Jersey USA). Recombinant protein purification and in vitro kinase assays All proteins were expressed and purified from E. coli strain BL21 or BL21-λP cells as recently described ( Dias et al ., 2023 ), using the constructs outlined in Supplemental Table S1 . In vitro kinase assays using Phospho-tag gel stain (APB Bio) were performed as per the manufacturer’s instructions and as previously described ( Bredow et al ., 2021 ). In vitro kinase assays using γP32-ATP were performed as recently described ( Dias et al ., 2023 ), using 2 μg kinase in a buffer containing 50 mM Tris-HCl (pH 8.0), 25 mM MgCl 2 and/or 25 mM MnCl 2 , 5 mM DTT, 5 μM ATP, and 0.5-2 μCi γP32-ATP. The buffer used in assays with His 6 -CPK28 contained 500 μM CaCl 2 and no MnCl 2 . Histone 3S from calf thymus was used as a universal substrate in some reactions (Sigma Aldrich H5505). All reactions were incubated for 60 minutes at 30°C. Reactions were stopped by adding 6× Laemmli buffer and heating at 80°C for 5 min. Proteins were separated by 10% SDS-PAGE. The gels were sandwiched between two sheets of transparency film, exposed to a storage phosphor screen (Molecular Dynamics) overnight, and visualized using a Typhoon 8600 Imager (Molecular Dynamics/Amersham). Gels were post-stained with Coomassie Brilliant Blue (CBB) R-250 (MP Biomedicals) or SimplyBlue SafeStain (Invitrogen; CBB G-250) and scanned. Phosphoproteomics In vitro kinase assays were performed using 2 μg His 6 -CPK28 as the kinase and 4 μg of either His 6 -MAZ K141N or His 6 -CARK7 K97N as substrates in a buffer containing 25 mM Tris-HCl (pH 8.0), 10 mM MgCl 2 , 100 μM CaCl 2 , 1 mM DTT, 1mM PMSF, and 100 μM ATP. Control reactions included His 6 -MAZ K141N or His 6 -CARK7 K97N with no kinase. All reactions were incubated for 60 minutes at 30°C and stopped by adding 6× Laemmli buffer and heating at 80°C for 5 min. Protein separation was carried out using 10% Mini-PROTEAN TGX Precast Gels (BioRad 456-1036), followed by post-staining with SimplyBlue SafeStain (Invitrogen; CBB G-250). Bands corresponding to the size of His 6 -MAZ K141N or His 6 -CARK7 K97N were excised using sterile razors, and washed with ethanol and sterile ddH 2 0. SDS-PA gel slices containing immobilized proteins were destained four times with 50% acetonitrile (ACN) in 100 mM tetraethylammonium bromide (TEAB) at 37°C for 10 min. Destain solution was removed and gel pieces were washed with 100 mM TEAB at 37°C for 10 min. Gel pieces were dehydrated by incubating in 100% ACN at room temperature (RT) for 10 min. Gel pieces were then fully dried at 37°C for 5 minutes. Cysteine residues were reduced by 10 mM dithiothreitol solution in 100 mM TEAB for 45 min at 37°C and then alkylated with 55 mM iodoacetamide in 100 mM TEAB buffer in the dark for 1 h at room temperature. Gel pieces were then washed in 50 mM TEAB buffer for 10 min, dehydrated by incubating twice in 100% ACN at RT and then fully dried at 37°C for 5 min. Gel pieces were rehydrated by adding 6 ng/μL trypsin (Promega SequencingGrade – V5113) in 50 mM TEAB. Peptides were digested for 16 h at 37°C shaking at 150 rpm. In-solution tryptic peptides were retained in a separate tube, and in-gel digested peptides were further extracted by adding 1% formic acid, 2% acetonitrile in 100 mM TEAB and allowed to incubate for 1 h at 37°C. This was followed by a second 1 h 37°C extraction using a 1:1 mixture of 1 % formic acid in 50 mM TEAB and 100% acetonitrile extraction buffer. This secondary tryptic peptide extraction fraction was pooled with the aforementioned retained in-solution tryptic peptides. Pooled peptides were then dried, re-suspended, and desalted using ZipTip C18 pipette tips (ZTC18S960; Millipore), as previously described ( Uhrig et al ., 2019 ). Desalted peptides were then dried and re-suspended in 3% (v/v) ACN / 0.1% (v/v) formic acid immediately prior to MS analysis. Data was acquired using a Thermo Fisher Scientific Orbitrap Fusion Lumos Tribrid system. Peptides were eluted into the mass spectrometer using an nLC-1200 (Thermo Fisher Scientific) mounted with an ES901 column (Thermo Fisher Scientific). Peptides were eluted using a 66 min gradient of increasing Buffer B 0 – 18% (3 min), 18 – 28% (14 min), 28-46% (11 min), 46-100% (5 min); Buffer A: 3% ACN, 0.1 % FA; Buffer B: 80 % ACN, 0.1 % FA. All mass spectra were acquired in data dependent acquisition mode. MS1 data were acquired using a resolution of 30000, a scan range of 375-1700 m/z, a maximum injection time of 50 ms and a RF lens setting of 30%. MS2 data were acquired using the ion trap in rapid scan rate mode, with maximum injection time set to dynamic. A 30 % HCD collision energy was using for peptide fragmentation. Subsequently, all data was analyzed using MaxQuant 2.0.3.0 ( Cox and Mann, 2008 ) using default parameters and the Araport 11 database ( Cheng et al ., 2017 ) with decoy mode set to revert. In brief, data search parameters included: trypsin cleavage permitting 2 missed cleavages, carbamidomethylation of cysteine residues (fixed modification), while methionine oxidation and phosphorylated serine/threonine/tyrosine were set as variable modifications. A PSM, peptide and protein FDR threshold of 0.01 was employed. Immunoblotting Proteins were separated by 10% SDS-PAGE, transferred onto polyvinylidene difluoride membranes (BioRad), and blocked with 5% nonfat milk or bovine serum albumin (BSA) in Tris-buffered saline containing 0.05–0.1% Tween-20. MAZ-GFP and MAZ K141N -GFP accumulation blots were probed with mouse anti-GFP (1:5,000, Roche 1814460001) and goat anti-mouse-HRP (1:10,000, Sigma A0168). MAPK activation blots were probed with rabbit anti-p44/42 MAPK (Erk1/2) (1:2,000, Cell Signaling 9102S) and goat anti-rabbit-IgG (1:10,000, Sigma A0545). Membranes were incubated with ECL Clarity Substrate (BioRad), visualized on a ChemiDoc Touch Imaging System (BioRad) and subsequently stained with Coomassie Brilliant Blue (CBB) R-250 (MP Biomedicals) as a loading control. Statistics GraphPad Prism 8 was used to perform statistical tests on all quantitative data, as indicated in figure legends. Data Availability All raw proteomic data have been uploaded to ProteomeXchange ( http://www.proteomexchange.org/ ) via the Proteomics IDEntification Database (PRIDE; https://www.ebi.ac.uk/pride/ ). Project Accession: PXD052679. Username: reviewer_pxd052679{at}ebi.ac.uk; Password: hAT8rFqbBmdt Author Contributions MGD and TD performed the majority of the work. MGD, TD, CGF, and JEM generated materials, performed experiments, and analyzed results. KRS and VNM generated materials. MCRG performed phosphoproteomics, supervised by RGU. MB and RD performed supporting experiments that are not shown, and RD curated phosphosites from online databases. Individual credits are included wherever possible in the figure captions and table legends. RS supervised JEM. JM designed the project, guided the work, supervised MGD, TD, CGF, MB, KRS, VNM, and RD, secured funding, and wrote the paper with input from all authors. Funding This work was funded by the following grants awarded to JM: Canadian Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery and Discovery Accelerator Programs [grant numbers RGPIN-2016-04787 and RGPAS-492902-2016], and the Canada Research Chair (CRC) Program [JM is CRC-II in Plant Immunology]; as well as the following grants awarded to RGU: Canadian Foundation for Innovation John R. Evans Leaders Fund (CFI-JELF; 41831 & 37833); and also the following grants awarded to RS: CEPLAS (EXC2048) and research group CSCS (FOR5235) of the German Research Foundation (DFG), which supported JEM. MGD was supported by a Research Internship Abroad Fellowship (BEPE) from the São Paulo Research Foundation (FAPESP) [grant number 2021/06835-3]. CGF was supported by an Ontario Graduate Scholarship (OGS 2019-2020). MB was supported by an NSERC Postdoctoral Fellowship (2019-2021). KRS was supported by an NSERC Undergraduate Summer Research Award (USRA 2017), NSERC Canada Graduate Scholarship (CGS-M 2017-2018) and an Ontario Graduate Scholarship (OGS 2018-2019). Supplemental Data Supplemental Table S1 . Primers, clones, and germplasm used in this study. Download figure Open in new tab Supplemental Figure S1. MAZ and CARK7 associate with but do not phosphorylate CPK28. (A-B) Split-luciferase complementation assays with FER-nLuc or CPK28-nLuc and cLuc-MAZ (A) or cLuc-CARK7 (B) . Total photon counts are plotted as relative light units (RLU) after co-expression of the respective proteins in N. benthamiana . Experiments were performed by CGF and MGD; data presented here were collected by MGD from 3 independent experiments (n=18), and are plotted together, denoted by black, teal, and magenta dots. The association between CPK28 and MAZ or CARK7 are significantly different from each control (Student’s unpaired t-test; p<0.0001). (C-D) In vitro kinase assays using His 6 -MAZ (C) or His 6 -CARK7 (D) as the kinase and catalytically-inactive His 6 -CPK28 D188A as the substrate, as visualized by Phostag staining. Protein loading is indicated by post-staining the membranes with Coomassie Brilliant Blue (CBB). Assays were performed more than 3 times each by TD with similar results; representative data are shown. Download figure Open in new tab Supplemental Figure S2. Phosphorylation sites identified on subgroup VIII-III RLCKs from various studies. (A) List of phosphorylation sites identified on subgroup VIII-III RLCKs from various phosphoproteomics studies, curated from online databases ( Durek et al ., 2010 ; Lin et al ., 2021 ) based on the following publications ( Reiland et al ., 2009 ; Durek et al ., 2010 ; Wang et al ., 2013 , 2018 b ; van Wijk et al ., 2014 ; Wu et al ., 2014 ; Roitinger et al ., 2015 ; Nukarinen et al ., 2016 ; Xiang et al ., 2016 ; Song et al ., 2018 ). (B) Multiple sequence alignment of subgroup VIII-III RLCKs with protein secondary structures indicated as visualized by ESPript ( Robert and Gouet, 2014 ). Residues highlighted in magenta correspond to those in (A) ; the magenta stars indicate highly conserved phosphorylation sites. Analysis done by RD. Download figure Open in new tab Supplemental Figure S3. Analysis of immune responses in maz-1; cark6-2 and cark7-1; cark9-1 mutants. (A-E) ROS production measured in relative light units (RLUs) after treatment with 100 nM flg22. Values represent means +/-standard deviation (n=4-6). Data presented in A-D was collected by CGF; data in E was collected by MGD. Lower-case letters indicate statistically significant groups determined by a one-way ANOVA followed by Tukey’s post-hoc test (p=0.01). These assays were repeated several times over a 5 year period by CGF, MGD, and JM; representative experiments are shown. (F) Western blot indicating the activation of MAPKs before (0 min) and after exposure to 200 nM flg22 (5, 10, 30 min) in the indicated genotypes. The anti-pERK1/2 antibody recognizes the phosphorylated/activated forms of MPK6, MPK3, and MPK4/11. Coomassie Brilliant Blue (CBB) staining of the same membranes indicates loading. Experiments were completed 4 times with similar results by MGD; representative data is shown. (G) Growth inhibition of entire 12 day-old seedlings grown in liquid 0.5x MS media supplemented with 100 nM flg22, relative to mean seedling weight of 12 day-old seedlings grown in liquid 0.5x MS media with no peptide. Data from 3 independent biological replicates performed by MGD are plotted together, denoted by black, teal, and magenta dots. A one-way ANOVA followed by Tukey’s post-hoc test indicates no significant differences between groups, indicated by lower case letters. Credits for genetic crosses and genotyping are provided in Supplemental Table S1 . Download figure Open in new tab Supplemental Figure S4. MAZ and CARK7 associate with themselves and each other. (A-B) Split-luciferase complementation assays with FER-nLuc or CARK7-nLuc and cLuc-MAZ (A) or cLuc-CARK7 (B) . Total photon counts are plotted as relative light units (RLU) after co-expression of the respective proteins in N. benthamiana . Experiments were performed by CGF and MGD; data presented here were collected by MGD from 3 independent experiments (n=18), and are plotted together, denoted by black, teal, and magenta dots. The association between CARK7-MAZ and CARK7-CARK7 are significantly different from the controls (Student’s unpaired t-test; p<0.0001). Download figure Open in new tab Supplemental Figure S5. Analysis of the DFG motif in subgroup VIII RLCKs from multiple species. Phylogeny of subgroup VIII RLCKs from Amborella trichopoda (3 members), Arabidopsis thaliana (11 members), Solanum lycopersicum (7 members), Glycine max (11 members), Cucumis sativus (3 members), Oryza sativa (10 members), Zea mays (13 members), Physcomitrium patens (2 members), and Marchantia polymorpha (1 member). A multiple sequence alignment was generated using MUSCLE in MEGAX ( Kumar et al ., 2018 ) and used to generate a neighbour-joining tree, visualized in iTOL ( Letunic and Bork, 2021 ). Proteins containing ‘DFN’ or ‘DFD’ motifs in place of the canonical ‘DFG’ motif are indicated by closed and open pink circles, respectively, while proteins containing motifs other than DFN or DFD are indicated by blank spaces. Proteins that have demonstrated autophosphorylation activity are indicated by closed blue circles, while those that are inactive are indicated by open blue circles. Proteins that have not yet been tested are indicated by blank spaces. Analysis done by JM. Acknowledgements We thank all members of the Monaghan Lab for their comments on this manuscript and for their commitment to fostering a welcoming and collaborative research environment. We thank Claire Wright for assistance with genotyping, Jack Moore for technical assistance and maintenance of the University of Alberta Mass Spectrometry and Proteomics Facility, Saeid Mobini for managing the Queen’s University Phytotron Facility, and the Center for Advanced Imaging (CAi) at Heinrich Heine University. 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Share Catalytically inactive subgroup VIII receptor-like cytoplasmic kinases regulate the immune-triggered oxidative burst in Arabidopsis thaliana Márcia Gonçalves Dias , Thakshila Dharmasena , Carmen Gonzalez-Ferrer , Jan Eric Maika , Maria Camila Rodriguez Gallo , Virginia Natali Miguel , Ruoqi Dou , Melissa Bredow , Kristen R Siegel , Richard Glen Uhrig , Rüdiger Simon , Jacqueline Monaghan bioRxiv 2024.05.30.596543; doi: https://doi.org/10.1101/2024.05.30.596543 Share This Article: Copy Citation Tools Catalytically inactive subgroup VIII receptor-like cytoplasmic kinases regulate the immune-triggered oxidative burst in Arabidopsis thaliana Márcia Gonçalves Dias , Thakshila Dharmasena , Carmen Gonzalez-Ferrer , Jan Eric Maika , Maria Camila Rodriguez Gallo , Virginia Natali Miguel , Ruoqi Dou , Melissa Bredow , Kristen R Siegel , Richard Glen Uhrig , Rüdiger Simon , Jacqueline Monaghan bioRxiv 2024.05.30.596543; doi: https://doi.org/10.1101/2024.05.30.596543 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Plant Biology Subject Areas All Articles Animal Behavior and Cognition (7644) Biochemistry (17726) Bioengineering (13916) Bioinformatics (42033) Biophysics (21486) Cancer Biology (18635) Cell Biology (25549) Clinical Trials (138) Developmental Biology (13397) Ecology (19940) Epidemiology (2067) Evolutionary Biology (24361) Genetics (15620) Genomics (22541) Immunology (17763) Microbiology (40468) Molecular Biology (17207) Neuroscience (88739) Paleontology (667) Pathology (2842) Pharmacology and Toxicology (4834) Physiology (7659) Plant Biology (15175) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9834) Zoology (2272)