S-acylation of P2K1 mediates extracellular ATP-induced immune signaling in Arabidopsis AbstractS-acylation is a reversible protein post-translational modification mediated by protein S-acyltransferases (PATs). How S-acylation regulates plant innate immunity is our main concern. Here, we show that the plant immune receptor P2K1 (DORN1, LecRK-I.9; extracellular ATP receptor) directly interacts with and phosphorylates Arabidopsis PAT5 and PAT9 to stimulate their S-acyltransferase activity. This leads, in a time-dependent manner, to greater S-acylation of P2K1, which dampens the immune response. pat5 and pat9 mutants have an elevated extracellular ATP-induced immune response, limited bacterial invasion, increased phosphorylation and decreased degradation of P2K1 during immune signaling. Mutation of S-acylated cysteine residues in P2K1聽results in a similar phenotype. Our study reveals that S-acylation effects the temporal dynamics of P2K1 receptor activity, through autophosphorylation and protein degradation, suggesting an important聽role for this modification in regulating the ability of plants in respond to external stimuli. IntroductionPlants are sessile organisms that rely exclusively on innate immunity to defend against detrimental microbes or pests, unlike mobile animals that possess adaptive immune systems. In order to perceive and trigger innate immune responses, plants employ a two-tier innate immune system, which includes critical plasma membrane (PM) localized receptors1. The first layer of defense is termed pattern-triggered immunity (PTI), since it relies on PM, pattern-recognition receptors (PRRs), which recognize conserved, pathogen-associated molecular patterns (PAMPs). Pathogens can also cause cell damage releasing damage-associated molecular patterns (DAMPs) that are also recognized by PM-localized, PRRs2. PTI can be defeated by pathogen-produced, effector proteins that target a variety of cellular components involved in innate immunity. Hence, the second layer of defense, termed effector trigger immunity (ETI), involves plant recognition systems that counter the action of these pathogen effector proteins3.PRRs encompass a variety of receptor-like kinase (RLK) subfamilies. Receptor-like kinases comprise a ligand-binding ectodomain, a transmembrane domain and an intracellular kinase domain. For example, FLS2 (FLAGELLIN SENSITIVE 2), containing a leucine rich repeat (LRR) ectodomain, recognizes bacterial flagellin via direct binding to a conserved 22-aminoacid epitope, flg224,5,6. Another well-studied plant LRR-RLK is the Arabidopsis EFR receptor, which recognizes EF-Tu via perception of the conserved N-acetylated epitope elf187,8. A second family of RLKs involved in PAMP recognition is the lysin motif (LysM-RLK) family, which includes CERK1 (CHITIN ELICITOR RECEPTOR KINASE 1). Chitin is a major constituent of fungal cell walls, which is directly bound by AtCERK1 (AtLYK1) and AtLYK5 in Arabidopsis9,10. Two plant LRR-RLK members, PEPR1 and PEPR2, were shown to specifically recognize AtPEP peptides released during cell/tissue damage11,12. Extracellular ATP (eATP) is also released during tissue damage or in response to specific elicitation, including pathogens13. A member of the lectin-RLK subfamily, P2K1 (DORN1), was shown to be the key receptor that recognizes eATP resulting in the induction of an innate immunity response14. P2K1 (LecRK-I.9) was identified as a positive regulator of plant defense against the oomycete pathogens, Phytophthora brassicae, Phytophthora infestans, and bacterial pathogen Pseudomonas syringae DC300015,16,17,18. On the other hand, eATP can elicit P2K1-mediated RBOHD phosphorylation to regulate stomatal aperture with important implications for regulating plant photosynthesis, water homeostasis, pathogen resistance, and ultimately yield19.While different PRRs recognize different PAMPs/DAMPs, the plant responses induced upon activation of these receptors are highly similar. For example, PTI is characterized by an elevation of cytoplasmic calcium (Ca2+), reactive oxygen species (ROS), nitric oxide (NO), the activation of mitogen-activated protein kinases (MAPKs), and the expression of immune related genes13,20. Activation of the PRR by its cognate ligand leads to autophosphorylation and transphosphorylation of other proteins that is often followed by receptor ubiquitination and degradation, which subsequently dampens the immune response21.In contrast to phosphorylation, other forms of protein covalent modification are poorly studied in plants, although they are known to occur. For example, S-acylation, a reversible acylation of cysteine residues via a thioester linkage, is well characterized in mammals but poorly studied in plants. Mammalian studies have shown a critical role for S-acylation in controlling PM association, subcellular trafficking, stability, protein-protein interactions, enzymatic activity, and many other functions22,23,24. Dynamic protein S-acylation is catalyzed by protein S-acyl transferases (PATs) that contain a conserved Asp-His-His-Cys (DHHC) catalytic domain, while deacylation is mediated by acyl protein thioesterases (APTs)24. In human, a family of 24 DHHC-PATs was identified and shown to be involved in many physiological processes, as well as diseases spanning from neuropsychiatric disorders to cancers, cellular differentiation, melanomagenesis, and so on24,25,26. Most human DHHC-PATs are localized to endomembrane compartments such as Golgi, endosomes, endoplasmic reticulum, with only two proteins, DHHC20 and DHHC21, found at that the PM, which mediate epidermal growth factor receptor (EGFR) signaling in cancer and inflammatory responses27,28.The genome of the genetic model plant, Arabidopsis thaliana, encodes 24 DHHC-PATs29. However, in contrast to humans, 12 Arabidopsis DHHC-PATs are localized to the PM, perhaps underlying the critical role that PM RLKs play in the ability of plants to respond to their environment24,29. There are relatively few studies describing the function of PATs in plants. Of the 24 encoded AtPATs, only seven have been studied and shown to play a role in root hair growth30,31, cell death, ROS production32,33, salt tolerance34, cell expansion and division35, male and female gametogenesis36, branching37, and early seedling growth and establishment38. Although a few plant proteins were previously shown to be S-acylated, including FLS239, specific substrates for these AtPATs are rare, while their biological functions or mechanisms are also unknown.In this manuscript, we provide evidence for a model in which the dynamics of autophosphorylation, S-acylation, and receptor turnover control the ability of the P2K1 receptor to elicit an immune response. In the presence of eATP, P2K1 rapidly autophosphorylates, with a concomitant accumulation in S-acylation, and, ultimately, turnover of the receptor from the PM. Ligand activation of P2K1 leads to phosphorylation of PAT5 and PAT9 that restores S-acylation of the P2K1 receptor, re-establishing the inactive, steady-state form of the receptor.ResultsPAT5 and PAT9 restrict the plant immune responseIn our previous study, we used a mass spectrometry-based in vitro phosphorylation strategy, termed kinase client assay (KiC assay)40, to identify putative substrates of the P2K1 kinase domain; such as the NADPH oxidase respiratory burst oxidase homolog D (RBOHD)19. In addition, this assay also identified two homologous genes, AtPAT5 and AtPAT9, which encode DHHC-PATs proteins likely involved in protein S-acylation (Fig.聽1a). Both PAT5 and PAT9 proteins were localized to the PM (Supplementary Fig.聽1a, b), consistent with possible interaction with PM-localized P2K1.Fig. 1: PAT5 and PAT9 control PTI response triggered by eATP.a Identification of PAT5 and PAT9 tryptic peptides as a substrate of P2K1 kinase by KiC assay. b Ligand-induced calcium influx. 5-day-old seedlings were treated with 100鈥壩糓 ATP for 15鈥塵in. RLU, relative luminescence units. Error bars indicate 卤SEM; n鈥?鈥? seedlings; *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01, P-values indicate significance relative to Col-0 and were determined by one-sided ANOVA with unpaired, two-tailed Student鈥檚 t test. c ROS production was measured in leaf disks after treatment with 200鈥壩糓 ATP纬S for 30鈥塵in. Leaf disks were taken from WT (Col-0), pat5, pat9, and pat5/9 double mutants, or their complemented lines PAT5 (NP::ATPAT5-HA/Atpat5) and PAT9 (NP::ATPAT9-HA/Atpat9). Error bars indicate 卤SEM; n鈥?鈥?2 leaf disks; *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01, P-values indicate significance relative to Col-0 with ATP纬S treatment and were determined by one-sided ANOVA with unpaired, two-tailed Student鈥檚 t test. d MAPKs activation of Arabidopsis leaf disks that treated with 200鈥壩糓 ATP纬S for the times indicated. Coomassie Brilliant Blue (CBB) staining of protein was used as loading control. e PAT5 and PAT9 negatively mediated bacterial invasion. Arabidopsis seedlings with the indicated genotype (x axis) were flood-inoculated with Pst. DC3000 bacteria and bacterial growth determined by plate counting 3 days post inoculation. Error bars indicate 卤SEM; n鈥?鈥?2 (biological replicates); means with different letters are significantly different (p鈥?lt;鈥?.05); P-values indicate significance relative to Col-0 and were determined by one-sided ANOVA with multiple comparisons and adjusted using Benjamini鈥揌ochberg post-test. Box extends from the 25th to the 75th percentile, whiskers denote minima and maxima (Boxplots, Col-0: max鈥?鈥?.76; min鈥?鈥?.88; center鈥?鈥?.82; Q2 (25%)鈥?鈥?.45; Q3 (75%)鈥?鈥?.43, pat5: max鈥?鈥?.76; min鈥?鈥?.45; center鈥?鈥?.62; Q2 (25%)鈥?鈥?.13; Q3 (75%)鈥?鈥?.05, pat9: max鈥?鈥?.97; min鈥?鈥?.3; center鈥?鈥?.47; Q2 (25%)鈥?鈥?.1; Q3 (75%)鈥?鈥?.71, pat5/9: max鈥?鈥?.55; min鈥?鈥?; center鈥?鈥?.9; Q2 (25%)鈥?鈥?.49; Q3 (75%)鈥?鈥?.21, PAT5-1: max鈥?鈥?.7; min鈥?鈥?.59; center鈥?鈥?.05; Q2 (25%)鈥?鈥?.76; Q3 (75%)鈥?鈥?.25, PAT5-2: max鈥?鈥?.64; min鈥?鈥?.85; center鈥?鈥?; Q2 (25%)鈥?鈥?.51; Q3 (75%)鈥?鈥?.15, PAT9-1: max鈥?鈥?.72; min鈥?鈥?.6; center鈥?鈥?.09; Q2 (25%)鈥?鈥?.58; Q3 (75%)鈥?鈥?.47, PAT9-2: max鈥?鈥?.7; min鈥?鈥?.78; center鈥?鈥?; Q2 (25%)鈥?鈥?.27; and Q3 (75%)鈥?鈥?.47). Experiments were repeated three times with similar results. f, g Ligand triggers PAT5 and PAT9 phosphorylation after treated with 200鈥壩糓 ATP, 1鈥壩糓 flg22, or 50鈥壩糶/ml chitin in their complemented lines PAT5 and PAT9. Lambda protein phosphatase (Lambda PP, 鈭?and +) was added to release phosphate groups. CBB was used as loading control. All experiments were repeated and analyzed three times with similar results.Full size imageTo determine whether PAT5 and PAT9 are involved in immune signaling by the P2K1 receptor, we investigated the phenotypes of pat5 or pat9 mutants聽by examining the response to ATP. Interestingly, the rapid elevation of cytoplasmic Ca2+ and ROS burst after ATP treatment were significantly increased in both mutants compared to the wild type (Col-0; Fig.聽1b, c and Supplementary Fig.聽1e). Given their possible roles in modulating plant receptor activity, we also tested these immune responses with other elicitors, including flg22 and chitin. Similar results were observed using flg22 or chitin as the elicitor (Supplementary Fig.聽1c, d, f, g), suggesting that PAT5 and PAT9 might impact other PRRs. Consistent with the increase in cytoplasmic Ca2+ and ROS, ATP, flg22, or chitin-triggered MAPKs activation was also higher in the pat5 and pat9 mutants relative to the Col-0 wild type, particularly at 30 and 60鈥塵in when wild-type MAPK activation was decreasing (Fig.聽1d and Supplementary Fig.聽1i, j). Consistent with these stronger immune responses, growth of the bacterial pathogen Pseudomonas syringae DC3000 (Pst. DC3000) after surface inoculation was significantly reduced in the pat5 or pat9 mutant plants (Fig.聽1e and Supplementary Fig.聽1h). These phenotypes were increased significantly in pat5/9 double mutant plants (Fig.聽1b, c, e, and Supplementary Fig.聽1c, d, f, g), indicating that the functions of PAT5 and PAT9 are partially redundant.In order to confirm PAT5 and PAT9 mediated PTI responses, we expressed the full-length PAT5 or PAT9 proteins, driven by their native promoters, in pat5 and pat9 mutants (Supplementary Fig.聽1k), respectively. Expression of these proteins fully complemented the pat5 and pat9 mutant phenotype, restoring a wild-type PTI response (Fig.聽1b, c, e and Supplementary Fig.聽1 c, d, f, g). Furthermore, we tested whether the activation of the PAT5 and PAT9 proteins was associated with these PTI responses. Indeed, addition of ATP, flg22, or chitin-triggered phosphorylation of PAT5 and PAT9 (Fig.聽1f, g and Supplementary Fig.聽1l). Taken together, the above findings reveal that PAT5 and PAT9 are functionally redundant and act to negatively regulate plant innate immunity mediated by PAMP/DAMP, including ATP, flg22, and chitin.The Arabidopsis PAT proteins are mainly clustered in three groups29, while PAT5, PAT6, PAT7, PAT8, and PAT9 are in the same clade and show high sequence conservation (Supplementary Fig.聽2a). We next examined their gene expression upon eATP and pathogen treatment. Quantitative RT-PCR (qRT-PCR) analysis showed that PAT5, PAT6, PAT9, and P2K1 were significantly upregulated upon ATP treatment after 30鈥塵in or 1鈥塰 compared with 0鈥塰 in wild type (Col-0; Supplementary Fig.聽2b). Meanwhile, the expression of PAT5 and P2K1 was also greatly increased 1 or 2鈥塰 after inoculation with Pst DC3000 (Supplementary Fig.聽2b). These data indicate that eATP or pathogen can induce PAT5 to PAT9 clade gene expression, suggesting they may be functionally redundant.PAT5 and PAT9 directly interact with P2K1To verify the KiC assay result that PAT5 and PAT9 are the phosphorylation substrates of the P2K1 receptor (ATP-elicited PTI), we used a variety of assays to look for protein-protein interaction. For example, split-luciferase complementation imaging (LCI) assays in Nicotiana benthaminana showed that both PAT5 and PAT9 can interact in vivo with unstimulated P2K1, but not RBOHC (Supplementary Fig.聽2c). Similarly, bimolecular fluorescence complementation (BiFC) assays in Arabidopsis protoplasts were used to specifically test for interactions at the PM. The yellow fluorescence signals were produced in the combination of PAT5/9-YFPC with P2K1-YFPN and merged well with the PM marker FM4-64 signal (Fig.聽2a). Interestingly, PAT5 and PAT9 appeared to form heterodimers and homodimers in both assays (Fig.聽2a and Supplementary Fig.聽2c). These results suggest that DAMP treatment may enhance the association between PAT5/PAT9 and P2K1 on the PM.Fig. 2: PAT5 and PAT9 directly interact with P2K1 in vitro and in vivo.a Interactions of PATs and P2K1 receptor at Arabidopsis protoplast plasma membrane. FM4-64 was used to stain the plasma membrane. Bar鈥?鈥?0鈥壩糾. b Directly interaction of PATs and P2K1 in vitro. Purified, recombinant proteins were incubated with Glutathione Sepharose 4B beads followed by GST-His pull-down assay. His-CD2b (At5g09390, CD2-binding protein-related, a substrate of P2K1 from KiC assay data) was used as a negative control. c PAT5 and PAT9 interact with P2K1 in vivo. The indicated constructs were transiently expressed in Arabidopsis wild-type protoplasts followed by Co-IP assays. All experiments were performed and analyzed three times with similar results.Full size imageThe LCI and BiFC results show that PAT5/PAT9 and receptors can form a complex, but do not indicate which domains are responsible for their interaction. Therefore, we performed in vitro pull-down assays using GST- or His-tagged recombinant proteins purified from E. coli. The PAT5 and PAT9 proteins are composed of four transmembrane domains (TMDs), the catalytic DHHC-Cysteine rich domain (DHHC-CRD) which is present in the cytosol and necessary for auto-acylation and for the modification of target proteins, and the N- and C-terminal cytoplasmic domains (Supplementary Fig.聽2d). Remarkably, PAT5-CRD, PAT5-C, PAT9-CRD, and PAT9-C were able to directly bind to C-terminal cytoplasmic domain P2K1-KD (Fig.聽2b). Meanwhile, an interaction with the kinase inactive form of the P2K1 kinase domain (P2K1-KD-1) was also observed in this pull-down assay (Fig.聽2b), suggesting that PAT5/PAT9-P2K1 interactions are independent of receptor autophosphorylation. Moreover, the PAT5-P2K1 and PAT9-P2K1 interactions were also detected in co-immunoprecipitation (Co-IP) assays when PAT5-Myc or PAT9-Myc were co-expressed with P2K1-HA in wild-type protoplasts (Fig.聽2c). Taken together, these various assays indicate that PAT5 and PAT9 directly interact with P2K1 in planta.P2K1 directly phosphorylates and stimulates PAT5 and PAT9We next sought to determine whether PAT5 and PAT9 are the direct phosphorylation substrates of P2K1 using kinase assays. Consistent with the KiC assay results, P2K1-KD strongly trans-phosphorylated PAT5-CRD, PAT5-C, PAT9-CRD, and PAT9-C, whereas the kinase dead P2K1-KD-1 failed to phosphorylate them (Fig.聽3a), indicating that P2K1 can directly phosphorylate PAT5 and PAT9.Fig. 3: P2K1 phosphorylates PAT5 and PAT9 to regulate immune responses.a P2K1 directly phosphorylates PAT5 and PAT9. Purified P2K1 kinase domain recombinant proteins were incubated with PAT5/PAT9-CRD and -C domains in an in vitro kinase assay. Autophosphorylation and transphosphorylation were measured by incorporation of 纬-[32P]-ATP. MBP and GST-CD2b were used as positive and negative controls, respectively. The protein loading was measured by CBB staining. Red stars represent the trans-phosphorylated proteins. Experiments were repeated two times with similar results. b, c Phosphorylation of PAT9 regulates ATP-induced calcium influx and ROS production. The indicated genomic PAT9 variants were transformed into the pat9 mutant, and then examined for ATP-induced calcium influx and ROS production for 30鈥塵in. RLU relative luminescence units. Error bars indicate 卤SEM; n鈥?鈥? (biological replicates); means with different letters are significantly different (p鈥?lt;鈥?.05); P-values indicate significance relative to Col-0 and were determined by one-sided ANOVA with multiple comparisons and adjusted using Benjamini鈥揌ochberg post-test. d PAT9 Phosphorylation mediates ATP-induced restriction of bacterial growth. The indicated plant leaves were syringe-infiltrated with 106 cfu/mL鈭? of Pst. DC3000 after 24鈥塰 water (mock) or 400鈥壩糓 ATP treatment, which is different from surface inoculation (Fig.聽1e). Bacterial numbers were determined 3 days post inoculation. Error bars indicate 卤SEM; n鈥?鈥?2 (biological replicates); means with different letters are significantly different (p鈥?lt;鈥?.05); P-values indicate significance relative to Col-0 with mock treatment and were determined by one-sided ANOVA with multiple comparisons and adjusted using Benjamini鈥揌ochberg post-test.Full size imageTo further explore the role of P2K1 in PAT9 phosphorylation during PTI in planta, we investigated the key phosphosites found by the KiC assay. We found that the phosphosites of PAT9 in the KiC assay were conserved in PAT9 orthologs or paralogs in different species, such as soybean, rice, and maize (Supplementary Fig.聽2e). We amplified the full-length genomic DNA of PAT9 with the native promoter, and substituted Thr107 and Ser109 with Ala (T107A/S109A), which blocks phosphorylation, or Asp (T107D/S109D), which mimics phosphorylation, and then transformed these constructs into pat9 mutant plants (Supplementary Fig.聽2d, f). The ATP-elicited cytoplasmic Ca2+ influx and ROS production in the T107A/S109A PAT9 variant plants were similar to the pat9 mutant plants and significantly higher compared to plants transformed with the WT PAT9 (Fig.聽3b, c), demonstrating that Thr107 and Ser109 are required for the activation of PAT9. Conversely, phospho-mimic mutations of T107 and S109 (T107D/S109D) were remarkably reduced in their ability to stimulate the ATP-elicited Ca2+ influx and ROS production (Fig.聽3b, c). To confirm the important role of these two phosphosites in mediating ATP-induced bacterial defense, we pretreated mutant leaves with water (mock) or 400鈥壩糓 ATP for 24鈥塰, and then syringe-infiltrated with 106鈥塩fu/mL鈭? of Pst. DC3000, which is different from the surface inoculation (Fig.聽1e), and then quantified bacterial numbers 3 days post inoculation. Relative to wild-type plants, the T107A/S109A variants showed greater resistance to bacterial growth, while the T107D/S109D phospho-mimic plants were more susceptible (Fig.聽3d). Based on these data, we proposed that T107 and S109 phosphosite variants appear to be partially functional. Meanwhile, to verify whether the key residue DHHC catalytic domain is responsible for PAT9 function, we mutated this sequence to DHHS (C166S) and expressed this variant in the pat9 mutant plants. The C166S PAT9 variant plants showed the same Ca2+ influx, ROS production and bacterial defense as the pat9 mutant (Fig.聽3b, d), demonstrating that the DHHC domain is required for the full function of the PAT9 protein.The above data, together with PTI-induced phosphorylation of the PAT5 and PAT9 proteins, indicate that P2K1 can directly phosphorylate and activate PAT5 and PAT9 in an ATP-induced manner, resulting in dampening of the ATP-induced PTI response including the rapid elevation of cytoplasmic Ca2+ and ROS production, and restriction of bacterial invasion.P2K1 is S-acylated by PAT5 and PAT9To investigate whether P2K1 can be S-acylated and which residues are targeted, we first predicted the potential S-acylation residues using GPS-lipid 1.0 software (online http://lipid.biocuckoo.org/webserver.php; Supplementary Fig.聽3). Based on these predictions, we generated a series of mutant forms of P2K1 in which cysteine (C) sites were individually mutated to serine (S). These mutant proteins were then transiently expressed in Arabidopsis protoplasts and their S-acylation status determined through a parallel S-acylation assay with or without the hydroxylamine thioester-cleavage step. Compared with the wild-type P2K1 form, the levels of S-acylation of single mutants such as P2K1C394S and P2K1C407S, but not P2K1C538S and P2K1C559S, were significantly reduced, while S-acylation of the double mutant P2K1C394/407S was below the level of detection (Supplementary Fig.聽4a). Remarkably, P2K1C394/407S protein migrated faster than the wild-type P2K1 when separated by SDS-PAGE (Supplementary Fig.聽4a). The substitution of C residue with a S residue could provide a potential phosphorylation site, and it is difficult to distinguish from phosphorylated P2K1. Therefore, we also replaced each C residue with alanine and transformed into p2k1-3 mutant plants (Supplementary Fig.聽4e). Consistent with C to S mutants, the levels of S-acylation of P2K1C394A, P2K1C407A and P2K1C394/407A were remarkably reduced (Fig.聽4a). Furthermore, loss of PAT5 and PAT9 function reduced P2K1 S-acylation levels, while complementation by expression of the wild-type PAT5 or PAT9 proteins restored these deficiencies (Fig.聽4b). However, the PAT5C188S and PAT9C166S mutants, in which the DHHC catalytic domain was mutated to DHHS, lost auto-S-acylation activity and failed to rescue P2K1 S-acylation levels (Fig.聽4b) when expressed in the pat mutant background. The above data indicate that PAT5 and PAT9 S-acylate P2K1 in planta.Fig. 4: PAT5 and PAT9 S-acylate P2K1.a Detection of P2K1 S-acylation and their associated residues in the stable P2K1-HA transgenic plants. The S-acylation levels were detected by an acyl-resin capture (acyl-RAC) assay. Palm- palmitoylated protein, Depalm- depalmitoylated protein. HAM, hydroxylamine, for cleavage of the Cys-palmitoyl thioester linkages. Experiments were repeated three times. b PAT5 and PAT9 S-acylate P2K1 in planta. Protoplasts of Arabidopsis wild-type Col-0, Atpat5 and Atpat9 mutants were transfected with plasmids as indicated, followed by acyl-RAC assays. Experiments were repeated three times. c鈥?b>e Dynamic S-acylation levels of P2K1 in stable transgenic plants upon eATP addition. The P2K1-HA transgenic plant was crossed with pat5, pat9, NP::PAT9-T107/AS109A/pat9, and NP::PAT9-T107D/S109D/pat9 plants and then infiltrated with 200鈥壩糓 ATP. All experiments were repeated and analyzed three times with similar results.Full size imageIn order to better examine the dynamic effects of S-acylation on the P2K1 receptor, we treated the stable transgenic P2K1-HA plant with eATP from 5 to 60鈥塵in. Under these conditions, S-acylation of P2K1-HA was remarkably increased 15 and 30鈥塵in after elicitation (Fig.聽4c). To investigate the relationship between S-acylation and phosphorylation of the P2K1 receptor, we treated plants with MG132, an inhibitor of proteasome-mediated protein degradation. Interestingly, we found that the dynamics of P2K1 S-acylation showed an inverse relationship to P2K1 autophosphorylation (Supplementary Fig.聽4b). Furthermore, the eATP-induced P2K1 S-acylation did not increase in the pat5 or pat9 mutants background (Fig.聽4d), revealing that PAT5 and PAT9 are required for the eATP-elicited P2K1 S-acylation. Additionally, the phospho-dead PAT9 variant PAT9T107AS109A plants failed to complement the eATP-induced P2K1 S-acylation, but phosphomimetic PAT9 variant PAT9T107DS109D plants showed stronger P2K1 S-acylation than wild type (Fig.聽4e). These results, together with above data, show that PAT5 and PAT9 S-acylate P2K1 in an ATP-induced and time-dependent manner, and P2K1 S-acylation requires phosphorylation of PAT5/9.Another important function of S-acylation is as a mediator of subcellular trafficking or membrane association, so we examined whether S-acylation regulates the localization of P2K1. First, we expressed GFP- and RFP-tagged fluorescent proteins in Arabidopsis protoplasts, the P2K1C394407S showed an identical PM localization as wild-type P2K1 (Supplementary Fig.聽4c), indicating that P2K1 is correctly trafficked to the plasma membrane in the absence of S-acylation. We also crossed 35S:P2K1-GFP plants with pat5 and pat9 mutant plants and found that the PM localization of P2K1 was not affected in both mutant backgrounds (Supplementary Fig.聽4d), suggesting that S-acylation is not involved in the subcellular targeting of the P2K1 receptor. S-acylation mediates P2K1 phosphorylation and turnover in a ligand-dependent mannerGiven that PAT5 and PAT9 directly interact with, are phosphorylated by and S-acylate P2K1 in a DAMP-dependent manner, we sought to determine whether the activation of P2K1 is regulated by PAT5 or PAT9. Previous results showed that autophosphorylation of P2K1 is induced by ATP elicitation19. Therefore, we expressed P2K1-HA in the Atpat9 and Atpat5 mutant background and found that, compared with P2K1-HA expressed in wild-type plants, the P2K1-HA/Atpat9 and P2K1-HA/Atpat5 plants showed greater P2K1 phosphorylation at 5, 15, and 30鈥塵in after ATP treatment (Fig.聽5a). The mobility of the phosphorylated protein was shifted by treatment with lambda protein phosphatase (lambda PP) to release any phosphate groups. The ubiquitin-mediated degradation of FLS2 is ligand, dose, and time dependent41. Similarly, the P2K1 protein was reduced to ~17% with 400鈥壜礛 ATP treatment for 1鈥塰, and this receptor turnover was also time dependent (Fig.聽5a, b, d). Moreover, loss of PAT9 or PAT5 in P2K1-HA/pat9 or P2K1-HA/pat5 plants significantly inhibited this ATP-induced P2K1 turnover (Fig.聽5a). On the other hand, the addition of 2-bromopalmitate (2-BP), a palmitate analog known to inhibit S-acyl transferase activity, increased P2K1 accumulation but not phosphorylation (Supplementary Fig.聽5a). These data demonstrate that PAT5 and PAT9 modulate activation of P2K1 protein in a ligand-induced manner, mediated by effects on both autophosphorylation and subsequent protein turnover.Fig. 5: PAT9 regulates ATP-induced P2K1 phosphorylation and turnover.a PAT5/9 controls P2K1 phosphorylation and turnover. P2K1-HA was analyzed by immunoblot in wild type and Atpat5 and Atpat9 mutants background upon addition of 400鈥壩糓 ATP. p-P2K1 phosphorylation of P2K1. Actin protein was used as loading control. Experiments were repeated three times with similar results. b鈥?b>d Analysis of turnover and phosphorylation of P2K1 proteins modified (C鈥夆啋鈥堿) at the site of S-acylation. The indicated constructs were expressed into Arabidopsis p2k1-3 mutant plant and treated with 400鈥壩糓 ATP. Lambda PP (-P) was added to release phosphate groups. In panel c and d, the ratio of P2K1 protein phosphorylation and total protein amount were measured from panel b and another replicate with Image J. Error bars indicate 卤SE; n鈥?鈥? (biological replicates). *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01, P-values indicate significance relative to WT and were determined by one-sided ANOVA with unpaired, two-tailed Student鈥檚 t test.Full size imageIn order to better understand how S-acylation mediates receptor activity upon elicitor stimulation, we examined mutated P2K1 protein in Arabidopsis with the substitution of C residue with alanine, but not a S residue, which may provide a potential phosphorylation site (Supplementary Fig.聽4a). Expression of the P2K1C394A and P2K1C407A protein showed more P2K1 protein phosphorylation but less protein turnover than the wild-type form upon ATP addition (Supplementary Fig.聽5b). Meanwhile, the double substitute forms of P2K1C394/407A protein also showed a significant reduction in ligand-induced protein turnover from 47% of wild-type levels remaining (26% in WT form) at 30鈥塵in to 33% (17% in WT form) at 1鈥塰 (Fig.聽5b, d and Supplementary Fig.聽5c). Furthermore, ATP-induced P2K1 protein phosphorylation increased to 45% at 5鈥塵in and then decreased to 6% at 1鈥塰 (Fig.聽5a鈥揷). Meanwhile, the P2K1C394/407A protein phosphorylation was remarkably increased relative to the wild-type P2K1 protein from 55% (45% in WT) at 5鈥塵in to 15% (6% in WT) at 1鈥塰 (Fig.聽5b, c and Supplementary Fig.聽5c). In summary, these results further demonstrate that S-acylation of key residues of the P2K1 protein plays an important role in mediating ligand-induced autophosphorylation and protein turnover.P2K1 S-acylation regulates PTIIn order to demonstrate the biological relevance of P2K1 S-acylation mediated by PAT5 and PAT9, we generated stable transgenic plants expressing the mutated S-acylation site proteins in the p2k1-3 mutant background using the full-length genomic sequences with a 1.5-kb native promoter (Supplementary Fig.聽5d). Similar to the data derived using the Atpat5 and Atpat9 mutant plants, ligand-induced elevation of cytoplasmic calcium levels in plants expressing P2K1C394A, P2K1C407A or P2K1C394/407A variants were significantly higher than similar mutant plants complemented with the wild-type P2K1 (Fig.聽6a). The ATP-induced resistance to the virulent bacterium Pst. DC3000 was also significantly enhanced in plants expressing P2K1C394A, P2K1C407A, or P2K1C394/407A compared to the corresponding wild-type controls (Fig.聽6b). In summary, consistent with both the in vivo and in vitro biochemical measurements, S-acylation of P2K1 negatively regulates the elicitor mediated PTI responses.Fig. 6: S-acylation of P2K1 negatively regulates PTI.a, b S-acylation of P2K1 mediates ATP-induced calcium influx and bacterial defense. The indicated 5-day-old seedlings were treated with 100鈥壩糓 ATP for calcium influx in 15鈥塵in. For bacterial growth, the indicated plant leaves were pretreated with 400鈥壩糓 ATP for 24鈥塰, and then inoculated with Pst. DC3000 and bacterial growth measured by plate counting after 3 days post inoculation. Values represent the mean鈥壜扁€塖EM, n鈥?鈥? in a, n鈥?鈥?2 in b (biological replicates); Means with different letters are significantly different (p鈥?lt;鈥?.05); P-values indicate significance relative to Col-0 with mock treatment and were determined by one-sided ANOVA with multiple comparisons and adjusted using Benjamini鈥揌ochberg post-test. c Model for the role of P2K1 receptor S-acylation in eATP signaling. Upon addition of the activating ligand eATP, the P2K1 receptor is rapidly autophosphorylation and phosphorylates downstream targets, leading to PTI immune response and P2K1 protein turnover. P2K1 directly interacts with and phosphorylates PATs to activate PATs S-acylation upon ATP treatment. Activation of PATs S-acylates P2K1 and then inactivates P2K1 phosphorylation and turnover, following dampens the immune response to protect plant growth.Full size imageDiscussionUnlike animals, plants are unable to move in response to danger or stress nor do they possess humoral and cellular immunity. Instead, plants have evolved an extensive and complex system of receptors that recognize pathogens, pests, and cellular damage through a large and diverse family of PM-localized RLKs. Ligand activation of these receptors leads to autophosphorylation, transphosphorylation of downstream proteins, and other cellular events culminating ultimately into a robust innate immunity response42. Concomitant with these events, the receptor is rapidly turned over through endocytosis, presumably to dampen the immune response that, while protective, is also detrimental to overall plant growth. Although each PRR recognizes a specific PAMP/DAMP signal, in general, the downstream signaling responses are highly similar regardless of which PRR is activated.Dynamic S-acylation determines protein function by influencing their association with membranes, compartmentalization in membrane domains, trafficking, and stability23. Our current study demonstrates that plant PRR activation is mediated by specific PAT proteins on the PM through S-acylation. Ligand activation of the P2K1 receptor leads to rapid autophosphorylation, downstream signaling, receptor turnover and, within 30鈥塵in restoration of S-acylation. Importantly, ATP-induced P2K1 S-acylation shows a clear inverse relationship with P2K1 autophosphorylation. Our hypothesis is that, S-acylation acts as a negative regulator of innate immunity, insuring a steady-state inactive state for P2K1 that insures against spurious activation, which might be detrimental to growth. Consistent with this model (Fig.聽6d), treatment with inhibitors of S-acylation or studies using mutants defective in PAT activity (pat5/pat9) or receptors mutated in their specific S-acylation sites all led to a marked elevation of the innate immunity response whether measured using cellular assays (e.g., cytoplasmic calcium levels) or pathogen sensitivity. A similar dynamic of receptor post-translational modification also occurs during activation of the epidermal growth factor receptor (EGFR) in human tumorigenesis and cancer progression28. Hence, at least for these specific examples, the 鈥榶in-yang鈥?between S-acylation and phosphorylation/turnover is common across plants and animals.Previous phylogenetic analysis revealed that the Arabidopsis PAT proteins were mainly clustered in three clades29. PAT5 and PAT9 belong to the largest and most homogenous group, and show sequence conservation with PAT6, PAT7, and PAT8. Our study reveals that PAT5 and PAT9 negatively control plant innate immunity mediated by PAMP/DAMP, and are, at least partially, functionally redundant. Meanwhile, we find that the claimed phosphosites of PAT9 are highly conserved in PAT6 and PAT8 in Arabidopsis, or orthologous genes in rice, soybean, and maize (Supplementary Fig.聽2e). Based on these data, we propose that PAT5/6/7/8/9 clade may be functionally redundant and similar mechanisms could apply to species other than Arabidopsis.In summary (Fig.聽6d), under steady-state conditions, the plant PRR P2K1 is S-acylated and distributed into the PM in an inactive state. In response to extracellular ATP binding, the P2K1 receptor was deacylated, which occurs concomitantly with P2K1 autophosphorylation. This activation ultimately leads to receptor endocytosis and ubiquitin-mediated protein turnover. PRR activation triggers downstream innate immune signaling, including rapid elevation of calcium influx, ROS, MAPKs activation, and other events leading to a robust innate immunity response. Meanwhile, activated P2K1 directly phosphorylates and stimulates PAT5 and PAT9, which will then S-acylate P2K1 receptor through the DHHC catalytic domain. In the case where the P2K1 receptor is not degraded, S-acylation is restored as an alternative means to dampen the immune response. These results are important since they clearly demonstrate how S-acylation mediates plasma membrane machinery and signaling during plant innate immunity, drawing clear parallels with well-studied signaling systems in animals. Missing steps include identification of the specific thioesterases that mediate the rapid deacylation and a clearer understanding of the full dynamics of the complex interactions within the receptor complex at the PM. Hence, much remains to be discovered about how plants, as well as other organisms, are able to survive in the face of the various pathogens or increasing severity of environmental change through the PM receptor complex.MethodsPlant materials and growth conditionsAll Arabidopsis thaliana plants used in this study are derived from the Columbia (Col-0) ecotype and express an T-DNA carrying aequorin43, including p2k1-3 (Salk-042209), pat5 (GABI-322D08), pat9 (SALK-003020C), and pat9-2 (SALK_206051C). Additional transgenic lines in this study are described below. Plants were grown in soil or 1/2 MS medium containing 1% sucrose at 21鈥?3鈥壜癈, 60鈥?0% humidity in a growth chamber under long day (16鈥塰 light/8鈥塰 dark) conditions.Constructs and transgenic plantsFull-length CDS or genomic DNA of P2K1 (At5g60300), PAT5 (At3g48760), PAT9 (At5g50020), RBOHC (AT5G51060), and CD2b (At5g09390), as well as their kinase domain or C-terminal domain, were amplified from wild-type plants using gene-specific primers (Supplementary Table聽1). The PCR products were cloned into pDONR-Zeo or pGEM-T Easy vectors. The different mutant forms were generated by PCR-mediated site directed mutagenesis.In order to generate constructs for the LCI assays in tobacco leaves, the full-length DNA from the pDONR-Zeo vector were cloned into pCAMBIA1300-Nluc and pCAMBIA1300-Cluc using LR cloning. For BiFC assays, we used pAM-PAT-35SS::YFP:GW, pAM-PAT-35SS::YFPc:GW, and pAM-PAT-35SS::YFPn:GW44 as the destination vectors to form fusions with split YFP at the C-termini of proteins in Arabidopsis protoplasts using LR cloning.To express specific proteins in Arabidopsis protoplasts, different mutated forms of P2K1, PAT5, and PAT9 from the source pDONR-Zeo source vectors were cloned into pUC-GW14 and pUC-GW1719 vectors using LR cloning.In order to generate NP::ATPAT5/pat5 and NP::ATPAT9/pat9 stable complemented transgenic plants or different mutated variants, the coding sequences driven by their native promoters were PCR-amplified from wild-type genomic DNA or CDS from cDNA, and then cloned into pGWB1, pGWB5 and pGWB13 using LR cloning.In order to generate constructs used for expressing different mutant forms of P2K1, the full-length P2K1 genomic sequence including ~1.5鈥塳b native promoter was amplified from genomic DNA by PCR, and cloned into pGWB1, pGWB13, pGWB1645 using LR cloning, respectively.For constructs expressing recombinant proteins in E. coli, the DNA fragments of P2K1-KD cut with EcoRI and XhoI was inserted into pGEX-5X-1. To gain His-tagged constructs, DNA fragments of PAT5-CRD, PAT5-C, PAT9-CRD, and PAT9-C cut with BamHI and XhoI were cloned into pET28a, while CD2b using SacI and XhoI, respectively.Kinase client assay (KiC assay)The KiC assay, instrument and detailed search parameters were used as before19,40. More than 2100 peptides identified from phosphorylation sites taken from a number of studies were individually synthesized and then incubated with the purified, recombinant GST-DORN1-KD kinase domain followed by ATP addition. The peptide mixture was then analyzed using a Finnigan Surveyor liquid chromatography (LC) system attached to a LTQ Orbitrap XL ETD mass spectrometer. Two sets of empty vectors (GST and MBP) and two kinase-dead proteins, GST-P2K1-1 (D572N) and GST-P2K1-2 (D525N), were used as negative controls.Calcium influx assayBriefly, 5-day-old seedlings were individually incubated with 50鈥壩糽 of reconstitution buffer containing 10鈥壩糓 coelenterazine (Nanolight Technology, Pinetop, AZ), 2鈥塵M MES buffer (pH 5.7), and 10鈥塵M CaCl2 in the wells of a 96-well plate in the dark at room temperature overnight. The next morning, 50鈥壩糽 of treatment solution (concentration was double strength to give a set final concentration of 25鈥塵M MES and 100鈥壩糓 ATP (Sigma, A2383), 1鈥壩糓 flg22 (GenScript), or 50鈥壩糶/ml chitin (Sigma, C9752)) was added to each well, and the luminescence was monitored using a CCD camera (Photek 216; Photek, Ltd.). The luminescence was monitored about 15鈥塵in for ATP treatment, 30鈥塵in for flg22 and chitin treatments.Oxidative (ROS) burst assayLeaf disks were taken from 4- or 5-week-old plants and incubated with 50鈥壩糽 ddH2O in the wells of a 96-well plate in the dark overnight. The next day, 50鈥壩糽 2x chemiluminescent luminol buffer was added to each well to be a final concentration of 25鈥壩糓 luminol, 20鈥壩糶/ml horseradish peroxidase and 200鈥壩糓 ATP纬S (Sigma, A1388), 1鈥壩糓 flg22, or 50鈥壩糶/ml chitin. Luminescence was immediately monitored using a CCD camera (Photek 216; Photek, Ltd.) for 30鈥塵in.MAPK phosphorylation assayLeaf disks from 4- or 5-week-old plants were incubated in the ddH2O overnight at 23鈥壜癈, and then treated with 200鈥壩糓 ATP纬S, 1鈥壩糓 flg22, or 100鈥壩糶/ml chitin for 0, 15, 30, or 60鈥塵in. Total protein was extracted with extraction buffer containing 50鈥塵M Tris (pH 7.5), 150鈥塵M NaCl, 0.5% Triton-X 100, and 1x protease inhibitor for 30鈥塵in on ice. The extracted total proteins were separated by 10% (w/v) SDS-PAGE gel and detected by immunoblotting with anti-phospho-p44/p42 MAPK antibody (Cell Signaling, 9101, dilution, 1:1000).Bacterial growth assaysBacterial growth was performed using flood inoculation of seedlings46. Generally, 50鈥塵l of P. syringae pv. tomato DC3000 (5鈥壝椻€?06 colony-forming units (CFU) ml鈭?) bacterial suspension containing 10鈥塵M MES pH 5.7, 10鈥塵M MgCl2, 0.025% Silwet L-77 was dispensed into plates containing about 14-day-old seedlings for 2鈥?鈥塵in. For, ATP-induced bacteria invasion, 4- or 5-week-old plant leaves were pretreated with 400鈥壩糓 ATP, 10鈥塵M MES pH 5.7, for 24鈥塰, and then syringe-infiltrated with 106 cfu/mL of Pst. DC3000. After 3 days post inoculation47, either the leaves or the whole seedling without the root were ground in 10鈥塵M MgCl2, diluted serially, and plated on LB agar with 25鈥塵M rifampicin. Colonies (CFU) were counted after incubation at 28鈥壜癈 for 2鈥? days.Gene expression (qRT-PCR)RNA was extracted from seedlings or leaves using TRIzol Reagent (Invitroge) and the first-strand cDNA synthesis using M-MLV RT (Promega) according to the manufacturer鈥檚 instructions. qRT-PCR was carried on an Applied Biosystems QuantStudio 6 Flex (ABI). The relative gene expression was quantified using 螖螖Ct method and normalized to UBQ expression. The primers used in the study are in Supplementary Table聽1.Split-luciferase complementation imaging assayThe Agrobacterium tumefaciens (GV3101) cells containing the indicated constructs were infiltrated into 4-week-old leaves of N. benthamiana and then incubated at room temperature for 48鈥塰 before LUC activity measurement. In all, 1鈥塵M d-luciferin was sprayed onto the leaves and then kept in the dark for 5鈥?0鈥塵in to allow the chlorophyll luminescence to decay, the luminescence was monitored using a CCD camera (Photek 216; Photek, Ltd.). Arabidopsis protoplast isolation and transformationThe isolation and transfection of Arabidopsis protoplasts were performed as previously described19 and used for the various assays as indicated. Briefly, 2鈥塯 of 14 days old seedlings were sliced and mixed with 15鈥塵l TVL Solution (0.3鈥塎 sorbitol and 50鈥塵M CaCl2). In all, 20鈥塵l of Enzyme Solution was then added containing 0.5鈥塎 sucrose, 10鈥塵M MES-KOH pH 5.7, 20鈥塵M CaCl2, 40鈥塵M KCl, 1% Cellulase (Onozuka R-10), and 1% Macerozyme (R10). This enzyme solution containing protoplasts were filtered through a 75-mm nylon mesh after a gentle swirling motion at room temperature for 15鈥?8鈥塰. Next, the protoplasts were gently covered with 10鈥塵l W5 Solution (2鈥塵M MES pH 5.7, 154鈥塵M NaCl, 125鈥塵M CaCl2, and 5鈥塵M KCl) without disturbing the sugar content gradient, following centrifugation at 100脳g for 7鈥塵in. About 10鈥塵l of protoplasts were collected at the interface of Enzyme Solution and W5 Solution were transferred to a new tube. The protoplasts were then washed twice with 15鈥塵l of W5 Solution and centrifuged for 5鈥塵in at 60鈥塯. The pelleted protoplasts were resuspended in 1鈥?鈥塵l MMG Solution containing 4鈥塵M MES pH 5.7, 0.4鈥塎 mannitol and 15鈥塵M MgCl2. For DNA-PEG鈥揷alcium transfection, 10鈥?0鈥壩糶 (about 10鈥壩糽) of plasmid was added to 100鈥壩糽 protoplasts and mixed gently. An aliquot of 110鈥壩糽 of PEG solution was mixed with this DNA-protoplasts by gently tapping the tube, and then incubated at room temperature for 15鈥塵in. The transfection mixture was mixed with 450鈥壩糽 W5 solution to stop the transfection process, and centrifugated at 100脳g for 1鈥?鈥塵in. The pelleted protoplasts were resuspended with 1鈥塵l W5 solution. After incubation overnight in the dark at 23鈥壜癈, the protoplast solution was used for the various assays.Bimolecular fluorescence complementation assayN- and C-terminal YFP protein fusions plasmids were co-transformed into Arabidopsis protoplasts as described above and then incubated at 23鈥壜癈 in a growth chamber overnight at dark. The YFP fluorescence was observed using a Leica DM 5500B Compound Microscope with Leica DFC290 Color Digital Camera. For FM4-64 staining, 2鈥壩糓 FM4-64 was used and incubated for 5鈥塵in at room temperature (the plasma membrane stain).In vitro pull-downRecombinant proteins GST-P2K1-KD, GST-P2K1-KD-1, His-PAT5-CRD, His-PAT5-C, His-PAT9-CRD, His-PAT9-C, or His-CD2b were expressed in E. coli and affinity purified using Glutathione Resin (GenScript) and TALON庐 Metal Affinity Resin (Clontech), respectively. For pull-down, 5鈥壩糶 GST and His recombinant proteins were incubated with 25鈥壩糽 Glutathione Resin beads in the pull-down buffer containing 25鈥塵M Tris-HCl pH 7.5, 100鈥塵M NaCl, and 1鈥塵M DTT for 2鈥塰 at 4鈥壜癈. The beads were washed more than seven times with the washing buffer 25鈥塵M Tris-HCl pH 7.5, 100鈥塵M NaCl, and 0.1% Triton-X 100. The bund proteins were eluted with 25鈥壩糽 elution buffer containing 50鈥塵M Tris-HCl pH 7.5鈥?, 15鈥?0鈥塵M GSH for ~15鈥?0鈥塵in. The proteins were separated using SDS-PAGE gels and detected by immunoblotting using anti-His (Sigma, H1029, dilution, 1:1000) and anti-GST-Hrp (GenScript, A00130, dilution, 1:1000).In vitro phosphorylation assaysFor the in vitro kinase assay, 2鈥壩糶 of purified GST-P2K1-KD or GST-P2K1-KD-1 kinases were incubated with 1鈥壩糶 His-PAT5-CRD, His-PAT5-C, His-PAT9-CRD, His-PAT9-C, or His-CD2b as substrate in a 20-渭l reaction buffer containing 50鈥塵M Tris-HCl pH 7.5, 50鈥塵M KCl, 10鈥塵M MgCl2, 10鈥塵M ATP, and 0.25鈥壩糽 radioactive [纬-32P] ATP for 30鈥塵in at 30鈥壜癈. The reaction was stopped by 5鈥壩糽 of 5x SDS loading buffer. The proteins were separated by SDS-PAGE (10%), followed by autoradiography for 3鈥塰. The proteins within the gel were visualized by staining with Coomassie blue. Myelin basic protein (MBP) was used as a positive control.Co-immunoprecipitation assayTotal proteins were extracted from protoplasts or plant tissues with an extraction buffer containing 50鈥塵M Tris (pH 7.5), 150鈥塵M NaCl, 0.5% Triton-X 100, and 1 脳 protease inhibitor for 1鈥塰 on ice. The samples were centrifuged at 20,000脳g for 15鈥塵in at 4鈥壜癈, the supernatant was decanted and 1鈥壩糶 anti-Myc or 30鈥壩糽 anti-HA agarose was added to the supernatant and incubated for 4鈥塰 or overnight with end-over-end shaking at 4鈥壜癈. In all, 25鈥壩糽 protein A resin was added for 2鈥塰, spun down and washed seven times with extraction buffer. After washing, 25鈥壩糽 1x SDS-PAGE loading buffer was added and heated at 100鈥壜癈 for 10鈥塵in. The proteins were separated by SDS-PAGE and detected by immunoblotting with anti-HA-HRP (Roche, 12013819001, dilution, 1:3000), anti-Myc-HRP (Sigma, SAB4700447, dilution 1:3000). S-acylation assayThe S-acylation assays were performed as previously described48. Generally, Arabidopsis protoplasts transfected with HA-tagged proteins were homogenized in lysis buffer containing 50鈥塵M Tris (PH 7.5), 150鈥塵M NaCl, 0.5% Triton-X 100, and 1x protease inhibitor for 1鈥塰 on ice. After centrifuged at 20,000脳g for 15鈥塵in at 4鈥壜癈, 50鈥塵M N-ethylmaleimide was added to the supernatant for blocking free sulfhydryl groups, and proteins were then immunoprecipitated using Anti-HA-Agarose beads with end-over-end shaking at 4鈥壜癈 overnight. The next day, the beads were washed three times with lysis buffer and eluted in 100鈥壩糽 0.1鈥壩糶/渭l HA peptide for 15鈥塵in. The eluted proteins were divided into two equal portions: one treated with 1鈥塎 hydroxylamine and the other with 1鈥塎 Tris路HCl (pH 7.4; as a control) in the presence of activated thiol-Sepharose 4B. Two hours later, the sepharose beads were washed three times with lysis buffer without protease inhibitor at room temperature, and then resuspended in 1x protein loading buffer and heated at 100鈥壜癈 for 10鈥塵in. Western blots were performed using anti-HA-HRP (dilution, 1:3000).Quantification and statistical analysisStatistical analysis was performed in GraphPad Prism 8. Error bars in the figures are standard deviation (SD) or the standard error of the mean (SEM鈥?鈥塖D/(square root of sample size)), and number of replicates is reported in the figure legends. Statistical comparison among different samples was carried out by one-way ANOVA. Multiple comparison tests were corrected by controlling the false discovery rate (FDR) using Benjamini and Hochberg鈥檚 method. Samples with statistically significant differences (*p鈥?lt;鈥?.05 or **p鈥?lt;鈥?.01 as indicated in the figure legends) were marked with different letters (a, b, c etc.).Reporting summaryFurther information on research design is available in the聽Nature Research Reporting Summary linked to this article. 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USA 114, 13206鈥?3211 (2017).CAS聽 PubMed聽 Article聽 PubMed Central聽Google Scholar聽 Download referencesAcknowledgementsResearch reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (grant no. R01GM121445 to G.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the Next-Generation BioGreen 21 Program Systems and Synthetic Agrobiotech Center, Rural Development Administration, Republic of Korea (grant no. PJ01325403 to G.S.). Additional funding was provided within the framework of the 3rd call of the ERA-NET for Coordinating Action in Plant Sciences through NSF grant 1826803 (to G.S.). This work also supported by Chinese Universities Scientific聽Fund of China Agricultural University (No. 2020RC011 to D.C.), and the National Natural Science Foundation of China (32070285).Author informationAuthor notesNagib AhsanPresent address: Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, OK, USAAffiliationsState Key Laboratory of Agrobiotechnology, College of Plant Protection, China Agricultural University, Beijing, ChinaDongqin Chen,聽Fengsheng Hao聽 聽Huiqi MuDivision of Biochemistry, C.S. Bond Life Science Center, University of Missouri, Columbia, MO, USANagib Ahsan,聽Jay J. Thelen聽 聽Gary StaceyDivisions of Plant Science, C.S. Bond Life Science Center, University of Missouri, Columbia, MO, USAGary StaceyAuthorsDongqin ChenView author publicationsYou can also search for this author in PubMed聽Google ScholarFengsheng HaoView author publicationsYou can also search for this author in PubMed聽Google ScholarHuiqi MuView author publicationsYou can also search for this author in PubMed聽Google ScholarNagib AhsanView author publicationsYou can also search for this author in PubMed聽Google ScholarJay J. ThelenView author publicationsYou can also search for this author in PubMed聽Google ScholarGary StaceyView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsD.C., F. H., and H.M. designed and performed the experiments and wrote the manuscript. N.A. and J.J.T. performed and supervised the kinase client screen for kinase targets. G.S. and D.C. supervised the study and edited the manuscript. All authors discussed the results and commented on the manuscript.Corresponding authorsCorrespondence to Dongqin Chen or Gary Stacey.Ethics declarations Competing interests The authors declare no competing interests. Additional informationPeer review information Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. Peer review reports are available.Publisher鈥檚 note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary information CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.