Poltergeist-Like 2 (PLL2)-dependent activation of the wound response distinguishes systemin from other immune signaling pathways

Poltergeist-Like 2 (PLL2)-dependent activation of the wound response distinguishes systemin from other immune signaling pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Letter Poltergeist-Like 2 (PLL2)-dependent activation of the wound response distinguishes systemin from other immune signaling pathways Andreas Schaller, Rong Li, Fatima Haj Ahmad, Anja Fuglsang, Anke Steppuhn, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4919676/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Jul, 2025 Read the published version in Nature Plants → Version 1 posted You are reading this latest preprint version Abstract Systemin, the first signaling peptide identified in plants, mediates induced resistance against insect herbivores in tomato 1 . Initially, systemin was perceived as a hormone-like, long-distance messenger that triggers systemic defense responses far from the site of insect attack. It was later found to rather act as a phytocytokine, amplifying the local wound response for the production of downstream signals that activate defense gene expression in distant tissues 2 . Systemin perception and signaling rely on the systemin receptor SYR1 3 . However, the specifics of SYR1-dependent signaling and how systemin signaling differs from other phytocytokine signaling pathways remain largely unknown. Here, we report that systemin activates the poltergeist-like phosphatase PLL2 in a SYR1-dependent manner. PLL2, in turn, regulates early systemin responses at the plasma membrane, including the rapid inhibition of proton pumps through the dephosphorylation of their regulatory C-termini. PLL2 was found to be essential for downstream defense gene induction, ultimately contributing to insect resistance. Biological sciences/Plant sciences/Plant immunity/Pattern recognition receptors in plants Biological sciences/Plant sciences/Plant stress responses/Herbivory Biological sciences/Plant sciences/Plant signalling Biological sciences/Plant sciences/Plant stress responses/Wounding Biological sciences/Plant sciences/Plant stress responses/Biotic Figures Figure 1 Figure 2 Figure 3 Introduction Like other phytocytokines, the 18-amino-acid systemin peptide is derived from a larger precursor protein by proteolytic processing 4,5 . Rapid cellular responses to systemin include the extracellular alkalinization response, an increase in cytosolic calcium, depolarization of the plasma membrane, and the induction of an oxidative burst 3,6-8 . These early systemin responses resemble those triggered by other phytocytokines and are hallmarks of pattern-triggered immune signaling 9-12 . They depend on the systemin receptor SYR1 resembling many other peptide and pattern recognition receptors (PRRs) in the large family of leucine-rich receptor kinases (LRR-RKs) 3 . However, despite apparent similarities in perception mechanism and early signal transduction events, systemin induces distinct downstream responses including the activation of the octadecanoid pathway for the production of jasmonates that are responsible for the systemic induction of herbivore defense gene expression, rather than pattern-triggered immunity (PTI) 13 . To get insight into the specific signaling events downstream of the SYR1 receptor kinase, we used phospho-proteomics in a systemin-responsive tomato cell culture. In a previous study, we had compared the response of these cells to systemin and to an inactive control peptide 14 . Here, focusing on SYR1-dependent signaling events, we compared systemin-induced changes in protein phosphorylation in wild-type and syr1 mutant cell cultures, generated by biolistic transformation with a CRISPR/Cas9 genome editing construct (Supplementary Table S1). A rapid increase in medium pH, i.e. the systemin-induced alkalinization response, was observed for wild type but not for syr1 . Confirming the specific loss only of SYR1 function, the response to flg22 that depends on FLS2 as PRR was unaffected in syr1 cells (Fig. 1a). For phospho-proteomics, syr1 and wild-type cells were harvested in a time series after systemin treatment in six biological replicates (Extended Data Fig.1a, b). Microsomal membranes were isolated from each sample, digested with Lys-C and trypsin, enriched for phosphopeptides, and subjected to mass spectrometry (LC-MS/MS) in untargeted data-dependent acquisition mode. Quantitative information was obtained for 4804 phosphopeptide species matching 2155 different proteins. Principal component (PC) analysis of phosphosites that responded to systemin treatment (sites differentially phosphorylated at P < 0.05, comparing all time points to time 0) clearly separated wild-type and syr1 samples. 43.4% of the total variance is explained by differences between the two genotypes (Fig. 1b). The second PC separated wild-type according to sampling time reflecting systemin-induced changes in protein phosphorylation. In contrast, all syr1 samples clustered closely together indicating that there is no such systemin response in the receptor mutant (Fig. 1b). We then performed k-means clustering of phospho-peptide abundance profiles to compare dynamic changes in protein phosphorylation between the two cell cultures (Fig. 1d, Extended Data Fig. 2a). The largest difference was observed for clusters 2 and 3, characterized by a rapid and transient decrease in phosphorylation at 1 min after systemin treatment. Less than 20% (198) of the peptides in these clusters were from syr1 , compared to > 80% (1036) from the systemin-responsive wild type (Fig. 1c). We then checked whether these 1036 peptides show time-dependent changes in phosphorylation in syr1 cells and, if so, to which cluster they belong. We found them in all clusters except 2 and 3; none of them showed the systemin-induced drop in abundance at 1 min after treatment (Fig. 1e). The data indicate that the SYR1-mediated systemin response is characterized by rapid and transient de-phosphorylation of cellular proteins, implying the activation of protein phosphatases early in systemin signaling. Most of the protein phosphatases showing SYR1-dependent changes in phosphorylation belonged to the PPM/PP2C family, and the largest number of phosphosites differentially regulated after systemin treatment was observed for poltergeist-like phosphatases (PLLs; Extended Data Fig. 2b,c), representing clade C in the PP2C family 15 . We focused our attention on PLL2 (Solyc06g076100) for which all three detected phosphosites showed a cluster 2-type phosphorylation response (Extended Data Fig. 2d). PLL2 was transiently dephosphorylated after systemin treatment at three sites in its N-terminal domain. For Ser151, phosphorylation was found to be decreased, for Ser142 and Ser160 it was reduced to below detection limits at 1 min after systemin treatment (Fig. 1f). Addressing the effect of reversible phosphorylation on PLL2 activity, we performed site-directed mutagenesis to replace the three serines with phospho-mimetic aspartate, or non-phosphorylatable (phospho-dead) alanine residues. The wild-type enzyme (PLL2 WT ), as well as its phospho-mimic (PLL2 3D ) and phospho-dead (PLL2 3A ) variants were expressed in N. benthamiana leaves as GFP fusions and purified by immuno-precipitation (GFP-trap; Extended Data Fig. 3). Phosphatase activity of the recombinant proteins was analyzed in vitro as the amount of inorganic phosphate released from a synthetic phosphopeptide substrate. PLL2 3A showed three-fold higher phosphatase activity, while the phospho-mimic PLL2 3D mutant was significantly less active compared to the wild-type enzyme (Fig. 2a, b). The data indicate that PLL2 is activated by dephosphorylation in response to systemin in a SYR1-dependent manner. Activated PLL2 may regulate downstream proteins by dephosphorylation, thereby contributing to the transient drop in phosphosite abundance observed after systemin treatment. MapMan was used to functionally characterize potential PLL2 targets among cluster 2 and 3 proteins showing the systemin-induced dephosphorylation response. Among the MapMan bins significantly enriched in clusters 2 and 3 are ‚solute carrier-mediated transport’ (bin 24.2) and ‚solute primary active transport’ (bin 24.1) (Extended Data Fig. 4a), including two plasma membrane proton pumps, LHA1 (Solyc03g113400) and LHA4 (Solyc07g017780) as putative targets of PLL2 (Extended Data Fig. 4b). LHA1 was also suggested as a putative PLL target by correlation analysis of phosphorylation profiles in our previous study comparing phosphoproteomic responses to systemin and an inactive control peptide 14 . Here, we observed the dephosphorylation of LHA1 and of a second plasma membrane proton pump (LHA4) at 1 minute after systemin treatment. Transient dephosphorylation was more pronounced for LHA1 compared to LHA4 and it depended on SYR1 (Extended Data Fig. 4c, d), suggesting a role for LHA1 downstream of SYR1 in the systemin signaling pathway. Plasma membrane H + -ATPases have previously been implicated in systemin-signaling in tomato, and in wound signaling in general 16-18 . Reduced proton extrusion as a result of proton pump inhibition may contribute to extracellular alkalinization and membrane depolarization, that are both necessary and sufficient for the activation of downstream defense gene expression 16,19 . The activity of plasma membrane H + -ATPases is controlled by reversible phosphorylation, with the second-to-last C-terminal threonine residue as the main regulatory site 20,21 . Phosphorylation of this residue and subsequent binding of 14-3-3 proteins stimulates proton pump activity, while dephosphorylation inactivates the enzyme 22,23 . This regulatory threonine is the site that was found to be dephosphorylated in LHA1 and LHA4 in response to systemin treatment (Extended Data Fig 4c, d). This is also the site that was dephosphorylated by PLL2 in the synthetic phospho-peptide we used as the substrate in our in vitro activity assay (Fig. 2a, b). This assay showed that PLL2 dephosphorylates the regulatory phospho-threonine from the C-terminus of LHA1, with highest activity for the phospho-dead PLL2 3A mutant (Fig. 2a, b). The data sugest that systemin-induced dephosphorylation and activation of PLL2 contribute to the alkalinization response by inhibition of plasma membrane proton pump activity. To corroborate this hypothesis, we first tested whether PLL2 co-localizes with the proton pumps at the plasma membrane. A potential myristoylation site for plasma membrane targeting is tentatively predicted for PLL2 24 . Plasma membrane localization was indeed observed for PLL2 WT and also for the PLL2 3A and PLL2 3D variants transiently expressed in N. benthamiana leaves (Extended Data Fig. 5a). Interaction of PLL2 with the plasma membrane H + -ATPases LHA1 and LHA4 was confirmed in N. benthamiana by bimolecular fluorescence complementation (BiFC; Extended Data Fig. 5b), and in co-immunoprecipitation (Co-IP) experiments testing for pull-down of GFP-tagged PLL2 by the flag-tagged regulatory (R) domain of the proton pumps. This experiment confirmed interaction of PLL2 with LHA4, which was observed for all three PLL2 variants (Fig. 2c). Hence, the interaction with LHA4 does not seem to depend on the phosphorylation status of PLL2. In contrast, only the hyperactive PLL2 3A mutant was found to interact with the regulatory C-terminus of LHA1 (LHA1-R; Fig. 2d). For PLL2 WT , interaction with LHA1-R was observed only after systemin treatment, which is consistent with the systemin-induced dephosphorylation and activation of PLL2 (Fig. 2e). No interaction was observed in response to flg22 (Fig. 2e). The data indicate that PLL2 needs to be in its dephosphorylated/activated state to interact with LHA1, and they further suggest that the SYR1-dependent dephosphorylation and activation of PLL2 distinguishes systemin from flg22-induced signaling. To test whether PLL2 controls proton pump activity in vivo , we first used the yeast strain RS-72 which expresses the endogenous H + -ATPase, PMA1 , under the control of the GAL1 promoter and, therefore, requires galactose as the sole carbon source for growth. On media containing glucose, the endogenous proton pump is not expressed and growth depends on the activity of plasmid-borne plant H + -ATPases 25 . Both LHA4 and LHA1 supported yeast growth indicating that the two pumps are expressed in yeast and active (Fig. 2f, g). Binding of 14-3-3 proteins in a western blot overlay confirmed presence of the activating phospho-threonine at the regulatory C-termini of the proton pumps (Fig. 2h, i). Yeast growth and phosphorylation of LHA1 were suppressed upon co-expression of PLL2, and this inhibition depended on PLL2 activity: it was strongest for the hyperactive PLL2 3A mutant, weaker for PLL2 WT , and not observed for inactive PLL2 3D (Fig. 2g). The data support our hypothesis that the alkalinization response depends on the systemin-mediated dephosphorylation and activation of PLL2 which, in turn, dephosphorylates and thereby inactivates the proton pump LHA1. PLL2-mediated inhibition of LHA4, on the other hand, did not seem to depend on the phosporylation status of PLL2. Yeast growth was impaired and phosphorylation of LHA4 was inhibited by co-expression of all three PLL2 variants (Fig. 2f, h). This result is consistent with our Co-IP experiments, which showed interaction of the regulatory LHA4 C-terminus with wild-type PLL2 as well as the PLL2 3A and PLL2 3D mutants (Fig. 2c). Apparently, all three PLL2 variants are able to bind LHA4, irrespective of phosphorylation status, thereby preventing proton pump phosphorylation and activation, resulting in impaired yeast growth. Loss-of-function analysis, using tomato pll2 mutants generated by CRISPR/Cas9 genome editing (Extended Data Fig. 6) in root growth assays, corroborated PLL2-mediated regulation of proton pump activity and extracellular pH in planta . As a readout for proton pump activity, we grew seedlings on pH indicator plates and monitored the acidification of the growth medium. Medium acidification was stronger for pll2 than for wild-type seedlings (Fig. 3a). Consistent with enhanced proton pump activity and the ‘acid growth’ hypothesis 26 , root growth was increased in pll2 compared to wild type (Fig. 3b). The same assays were also used to test in planta , whether PLL2 is required for systemin-mediated inhibition of proton pump activity and seedling growth. In wild-type seedlings, systemin treatment resulted in extracellular alkalinization, and this response was lost in pll2 (Fig. 3c). Consistent with increased cell wall pH, seedling growth was inhibited by systemin treatment in both roots and hypocotyls. Growth inhibition was substantially reduced in pll2 compared to wild-type seedlings (Fig. 3d; Extended Data Fig. 7). The data confirm that systemin-induced pH responses depend on PLL2. The cumulative evidence supports our hypothesis that systemin signaling involves the SYR1-dependent activation of PLL2 which, in turn, dephosphorylates and thereby inactivates the proton pump LHA1 to result in the systemin-induced increase in extracellular pH and growth inhibition. We then tested whether PLL2 function is also required for downstream signaling and systemin-induced defense responses. The oxidative burst observed in wild-type plants in response to systemin treatment was clearly reduced in the pll2 mutant (Fig. 3e). Reconstituting systemin signaling in N. benthamiana , by co-expression of the SYR1 receptor with PLL2 or its phospho-mimic and phospho-dead mutants, we observed that the intensity of the systemin-induced ROS burst correlates with PLL2 activity. ROS production was highest for the hyperactive PLL2 3A compared to PLL2 WT and the inactive PLL2 3D mutant (Fig. 3f). Interestingly, the flg22-induced ROS burst was not affected by the tomato PLL2 variants suggesting differences in systemin and flg22-mediated regulation of ROS production (Fig. 3g). While the data do not preclude the possibility that PLL2 is involved in flg22 signaling as well, they indicate that the regulation of PLL2 activity by de-phosphorylation of serines 142, 151 and 160, is a specific feature of systemin signaling. In addition to the oxidative burst, the activation of mitogen-activated protein kinases (MAPKs), and the induction of defense gene ( PI-II ) expression in response to systemin treatment or wounding were also compromised in pll2 compared to wild type (Fig. 3h-k). As a result of impaired wound signaling, pll2 accumulated lower amounts of defensive proteinase inhibitors than wild-type plants when fed upon by larvae of the specialist herbivore Manduca sexta (Fig. 3l). Consequently, larvae gained weight more rapidly on pll2 mutants compared to wild type (Fig. 3m) indicating a loss of insect resistance. In this study, we identified PLL2 as an element of the SYR1-dependent wound signaling pathway for induced herbivore defense. SYR1-dependent activation of PLL2 by dephosphorylation at three regulatory sites is required for the systemin-induced alkalinization response, ROS burst and MAPK activation. These early cellular systemin responses are also hallmarks of PTI. Interestingly and in contrast to tomato PLL2, close homologs in Arabidopsis (AtPLL4 and AtPLL5) and rice (XB15) have previously been identified as negative regulators of PTI 27,28 . These PLL2 homologs interact with multiple PRRs to dampen PTI. Upon ligand perception, AtPLL4 and 5 are phosphorylated at some of the seven predicted phosphorylation sites in their regulatory N-termini (Extended Data Fig. 8) and dissociate from the receptor complex. Thereby inhibition is released and PTI activated 27 . We conclude that closely related PLLs act downstream of SYR1 as well as PRRs, but are differentially regulated after perception of the respective ligands. Dephosphorylation and activation of PLL2 is observed specifically in response to systemin and may explain the systemin-specific induction of herbivore resistance, while the activation of PTI involves the phosphorylation of AtPLL4/5 to release PRRs from AtPLL4/5-mediated inhibition. We further report that PLL2 affects the proton gradient across the plasma membrane and extracellular pH by dephosphorylation and inhibition of plasma membrane H + -ATPases. PLL2 targets the penultimate phospho-threonine residue in the regulatory C-terminal domain of plasma membrane proton pumps LHA1 and LHA4. The same site is also targeted by auxin signaling. However, in contrast to PLL2-mediated proton pump inhibition and extracellular alkalinization, auxin involves a different phosphatase (PP2C-D) and a pair of antagonistic kinases (TMK1 and TMK4) to increase phosphorylation of the regulatory threonine, thereby stimulating proton extrusion and acid growth 29-31 . In addition to the penultimate phospho-threonine residue, there are multiple other phosphosites in the regulatory C-terminus regulating proton pump activity, including serine 899. This serine is phosphorylated for proton pump inhibition in response to RALF and flg22 32,33 . Therefore, consistent with the specific role of PLL2 in systemin signaling, the regulatory mechanisms for proton pump inhibition appear to be different for the wound response and PTI. Methods Plant material and growth conditions. Tomato ( Solanum lycopersicum cv. UC82B) and N. benthamiana plants were cultivated in growth cabinets at 26 °C, with 16 h photoperiod, 100 µmol m -2 s -1 light intensity and 75% rel. humidity. For seedling growth assays, tomato seeds were sterilized in 2% [v/v] bleach with 3 drops of Tween 20 per 25 ml, and then washed in sterile ddH 2 O. For germination, seeds were placed on ATS medium 30 containing 1% [w/v] sucrose. After 1-2 days, seeds that had just germinated were transferred onto either ATS control plates, or ATS plates containing 100 nM systemin for another 5-7 days of growth under the same condition as for plants. The tomato ( Solanum peruvianum ) cell suspension culture was maintained as described 14,34 . For the syr1 mutant cell culture, the medium was supplemented with 75 mg/l kanamycin. Elicitor and wound ing treatments . Systemin (AVQSKPPSKRDPPKMQTD) and the SlLHA1 C-terminal Thr955 phosphopeptide (GLDIETIQQSYT (ph)V) were obtained from PepMic (Suzhou, China) at >95 % purity. Flg22 (QRLSTGSRINSAKDDAAGLQIA) was ordered at >95% purity from GenScript (USA). All peptides were dissolved in ddH 2 O at 1 mM and stored at -20 °C. For mechanical wounding, the first two true leaves of 3-week-old tomato plants were symmetrically squeezed with a hemostat from both sides of the midrib at each leaflet. Phosphoproteomics and data preprocessing . The setup of phosphoproteomics was adapted from Haj Ahmad et al. 14 . 200 ml cell culture (wild-type and syr1 ) was harvested at 0, 1, 2, 5, 15, and 45 min after addition of 10 nM systemin. Samples were collected from six independent batches of cells. Extraction of the microsomal fraction, phosphopeptide enrichment and LC-MS/MS were conducted as previously described 14 . Phosphosites were mapped against Solanum lycopersicum ITAG3.2 by MaxQuant version 2.4.2.0 35 . Overall 4804 phosphosites were obtained excluding hits to reverse sequences and potential contaminants, and sites with localization probability ≤ 0.75. Phosphosite intensities in the MaxQuant output table (Phospho (STY)Sites.txt) were normalized and log2 transformed in R 36 version 4.1.0 (all R scripts are available at GitHub ( https://github.com/shibalili ) under ‘systemin-project’). Data manipulation and visualization were done using tidyverse 37 and ggplot2 38 packages.  Exploratory data analysis. The R package Limma 39 was used to analyze differentially phosphorylated phosphosites. For each phosphosite, a linear model was fit to describe its phosphorylation profile over time. Coefficients for each time point were compared to time 0 in multiple pairwise comparison. Sites differentially phosphorylated relative to the same genotype–treatment combination at time 0 were identified at P < 0.05. Phosphosites which were differentially phosphorylated ( P < 0.05) in WT cell culture were considered for Principal Component Analysis (PCA) using the prcomp function in R. For analyses that require a complete set of data points for each phosphosite (e.g. k means clustering), missing values were imputed using the R package missForest 40 . A relatively loose cut-off of > 10 of maximum 72 data points per phosphosite was applied, which allowed 2450 (50 %) of the phosphosites with variable phosphorylation patterns to be retained for cluster analysis. Gap statistics 41 was applied to identify the optimal number of clusters as 5 (Extended Data Fig. 1b). Temporal phosphorylation profiles were clustered using the eclust function together with kmeans and euclidean distance from the R package factoextra 42 . MapMan enrichment analysis. Functional annotation of proteins was done using MapMan 43 ontology downloaded from MapManstore ( https://mapman.gabipd.org/mapmanstore ), and the Solanum lycopersicum genome annotation ITAG3.2 44 from PHYTOZOME V13.0 (https://phytozome-next.jgi. doe.gov/). Enrichment analysis was performed with the package hypeR 45 , using hypergeometric enrichment test to determine if a group of proteins is over-represented. Molecular Cloning. Polymerase chain reaction (PCR) primers for DNA amplification from plant genomic DNA or plasmid templates are listed in Supplementary Table 2. To generate expression clones for full-length wild-type SlPLL2 ( SlPLL2 WT ), a synthetic DNA fragment (Integrated DNA Technology, IDT) corresponding to the first 936 bp of PLL2 was inserted into pDONR221 by BP reaction (Invitrogen). The catalytic domain 14 was added by restriction enzyme cloning. To generate the SlPLL2 3A and SlPLL2 3D mutants, a Cla I(277)- Bcl I(509) fragment of SlPLL2 WT including serines 142, 151 and 160 was replaced with synthetic fragments in which the respective codons had been replaced by GCA for alanine, or GAT for aspartate, respectively. To generate SlPLL2 WT/3A/3D -sfGFP constructs for transient expression in N. benthamiana , the SlPLL2 WT/3A/3D variants and the coding sequence of sfGFP were amplified by PCR and inserted into pART7 between the CaMV 35S promoter and terminator. After Not I digestion, this expression cassette was transferred into pART27 46 . For the BiFC assay, the following fusion constructs were generated: SlLHA1-YFP, SlLHA1-nYFP, SlLHA4-YFP, SlLHA4-nYFP and SlPLL2-cYFP. To this end, the open reading frames (ORFs) of SlLHA1 (Solyc03g113400) and SlLHA4 (Solyc07g017780) were PCR-amplified from tomato root cDNA and introduced into the Xho I and Cla I sites of pART7. The ORFs for full-length EYFP (residues 1-240) and the N-terminal fragment of EYFP (nYFP, residues 1-155) were inserted as the Cla I- Xba I fragment downstream of SlLHA1/4 to result in the SlLHA1/4-YFP and Sl L HA1/ 4-nYFP fusions, respectively.Similarly, ORFs for SlPLL2 and the C-terminal fragment of EYFP (cYFP, residues 156-240) were fused to result in Sl PLL2-cYFP . For the Co-IP assay, the following expression constructs were generated: SYR 1 , 3xFLAG-LHA1-R and 3xFLAG- L HA 4 -R , in addition to Sl PLL2 WT -sfGFP described above. A genomicfragment comprising SYR1 (Solyc03g082470) was PCR-amplified from genomic DNA and cloned into pMB35S 47 using the Eco RV and Xba I sites. The regulatory domain of SlLHA1 (aa 864-956) and SlLHA4 (aa 859-952) were PCR-amplified from full-length cDNA templates. The 3xFLAG tag sequence was included in the PCR primers and added at the N-terminus of SlLHA1 and SlLHA4. The yeast complementation assay required expression vectors for Sl L HA 4 and SlLHA1 under control of the endogenous PMA1 promoter. For this purpose, the AtAHA2 ORF in the previously described pPMA1:AtAHA2 expression plasmid pMP1745 was replaced by Sl L HA 4 or SlLHA1 by Xho I- Nde I digestion 25 . Sl PLL2 and its phosphor-variants were cloned into the pMP1612 expression vector using Not I 25 . For CRISPR/Cas9-mediated genome editing, single guide RNA (sgRNA) constructs were generated in the pKSE401 binary vector comprising the pCaMV35S::Cas9 expression construct 48 . CRISPRdirect ( https://crispr.dbcls.jp ) was used to select guide RNAs that included a restriction site at the location of the double strand break to facilitate subsequent genotyping. sgRNA expression cassettes were generated by PCR using primers containing the sgRNA sequence and a Bsa I site for golden gate cloning, and pCBCT1T2 and/or pCBCT2T3 as the template. Primer sequences are listed in Supplementary Table 3. The resulting pKSE401-gSYR1 and pKSE401-gPLL2 editing constructs contained 2 and 3 sgRNAs for the genomic SlSYR1 and SlPLL2 loci, respectively. Plant transformation. CRISPR/Cas9 vectors were introduced into the cotyledons of 10-day-old etiolated tomato seedlings via Agrobacterium tumefaciens (GV3101)-mediated transformation. Transgenic lines were selected on media with kanamycin concentrations increasing successively from 35 mg/l to100 mg/l as described 49 . The syr1 mutant cell culture was also generated by CRISPR/Cas9 genome editing. The editing construct was linearized with Pme I and shot into S . peruvianum cells using the PDS1000/He Biolistic Particle Delivery System (BioRad) as described 50 . Transformed cells were selected on 75 mg/ml kanamycin for callus growth. Suspension cultures were established from calli that were confirmed by PCR to carry the editing construct and genotyped as follows. Genotyping of CRISPR/Cas9 mutants. Genomic DNA was isolated from WT tomato plants or S. peruvianum calli and corresponding CRISPR/Cas9 transformants. For transgenic plants, three independent syr1 and slpll2 mutants were identified by PCR and sequenced in the T0 generation (primers for genotyping are listed in Supplementary Table S3). Homozygous mutants from the segregating Cas9-free T2 progeny were used for experiments (Supplementary Table S1). For transgenic cell cultures generated by particle bombardment, genotyping was done by cloning the PCR-amplified target loci in pCR2.1-TOPO (Thermo Fisher Scientific). Eight clones per cell culture were sequenced revealing different mutations at the target site of the sgRNA and confirming absence of the wild-type sequence (Supplementary Table S1). RNA isolation and Reverse Transcriptase (RT)-qPCR. Total RNA was extracted from leaves of 3-week-old tomato plants with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA concentration was measured at 260 nm using a microplate reader (Tecan Spark) after DNase I digestion (Thermo Scientific). One μg RNA was reversely transcribed to synthesize the cDNA with RevertAid reverse transcriptase (Thermo Fisher Scientific) and oligo(dT) primers. Real-time PCR (qPCR) was performed in a Bio-Rad CFX Connect real-time PCR instrument (Bio-Rad; Munich, Germany) with SYBR-Green (Cambrex Bio Science Rockland Inc.; Rockland, ME, USA). RT-qPCR primers are listed in Supplementary Table S4. Target gene expression was analyzed by delta-delta Ct method and normalized to the expression level of three house-keeping genes, UBQ10 , ACTIN2 and  EF1 α . Transient protein expression in N. benthamiana . Transient expression experiments were performed essentially as described 51 . Briefly, agrobacteria carrying binary vectors with expression constructs for the protein(s) of interest and the P19 silencing suppressor were mixed to result in OD 600 = 0.5. When PLL2 mutants were expressed for activity assays, the OD 600 ratio (PLL2 WT/3A/3D or free GFP : P19) was 9:1. N. benthamiana lacks a functional systemin perception system. Therefore, in order to reconstitute systemin signaling in N. benthamiana , a SYR1-eGFP expression construct kindly provided by Georg Felix (University of Tübingen) was always co-infiltrated with the PLL2 constructs (PLL2 WT/3A/3D : SYR1 : P19 = 8:1:1). For Co-immunoprecipitation, the OD 600 ratio was PLL2 WT/3A/3D : LHA1/4 : P19 = 4.5:4.5:1, or PLL2 WT : LHA1 : SYR1 : P19 = 4:4:1:1.The infiltration buffer contained 10 mM MgCl 2 , 10 mM MES, pH 5.6 and 150 μM acetosyringone. Leaves were harvested 3 days post infiltration. Alkalinization assay. Medium alkalinization in response to systemin (10 nM), flg22 (20 nM) and ddH 2 O as the control was analyzed in S. peruvianum wild-type and syr1 cell suspensions seven days after subculture as described previously 34 . ROS burst. ROS production in response to systemin (30 nM), flg22 (30 nM) and ddH 2 O as the control was analyzed in 4 mm leaf discs of 4-week-old tomato or N. benthamiana plants as described previously 52 . MAPK activation. Three-week-old tomato plants with two true leaves were dipped briefly into 1 μM systemin in 0.05% [v/v] silwet-77 or the solvent alone, and at the indicated time points, leaf tissue was harvested. 100 mg tissue samples were ground in 100 μl extraction buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.5% [v/v] Triton X-100, 10 mM ß-mercaptoethanol, 1:1000 Protease Inhibitor Mix P (SERVA), 1:2000 Phosphatase Inhibitor Cocktail 2 and 1:2000 Phosphatase Inhibitor Cocktail 3 (Sigma)) using a bead beater (TissueLyzer LT; QIAGEN). The supernatant was collected after centrifugation at 16100 g for 10 min at 4 °C. Protein samples were mixed with SDS loading buffer, denatured at 95 °C for 5 min, separated by 10% [w/v] SDS–PAGE, and blotted to a nitrocellulose membrane. Western blots were developed to detect phosphorylated MAPKs using anti-pERK1/2 (1:5000; Cell Signaling) as the primary and goat anti-rabbit IgG (1:10000; Millipore) as the secondary antibodies, followed by enhanced chemiluminescence detection in a LICORbio Odyssey XF imager. In vitro phosphatase assay. The pART27-based expression constructs harboring phosphatase (SlPLL2 WT , SlPLL2 3A and SlPLL2 3D )-sfGFP were agro-infiltrated into N. benthamiana leaves. Three days post-infiltration, 100 mg leaf samples were harvested and ground in 100 μl extraction buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.5% [v/v] Triton X-100, 10 mM ß-mercaptoethanol, 1:1000 Protease Inhibitor Mix P) using a bead beater (TissueLyser LT; Qiagen). The extracts were cleared by centrifugation. An aliquot of the supernatant was used for the quantification of phosphatase expression levels by anti-GFP Western Blot analysis. The remainder was subjected to GFP-trap (Chromotek) to immuno-precipitate SlPLL2 variants according to the manufacturer’s instructions. On-bead phosphatase activity assays were performed using the Serine/Threonine Phosphatase Assay Kit (Promega), with the bead volume adjusted to result in equal amounts of enzyme for each of the PLL2 variants (Extended Data Fig. 3). The phospho-peptide GLDIETIQQSYT (ph)V, custom-synthesized at >95 % purity (PepMic; Suzhou, China), was used as the substrate at 100 µM. Co-immunoprecipitation. Constructs bearing phosphatase-sfGFP fusion constructs and the 3xFLAG-tagged regulatory domains of LHA1 and LHA4 were transformed in A. tumefaciens and co-infiltrated in N. benthamiana leaves in the combinations indicated. For elicitor treatments, the leaves co-expressing phosphatases and proton pumps were infiltrated with 100 nM systemin or 100 nM flg22, harvested and flash-frozen in liquid nitrogen within 2 min. Two infiltrated leaves (~2 g) were ground in 2 ml cold IP buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 % [v/v] glycerol, 5 μM DTT, 0.5% [v/v] IGEPAL, 1 mM PMSF, 1:1000 Protease Inhibitor Mix P from SERVA) on ice. The extract was cleared by centrifugation (16100 g, 15 min, 4 °C) and 50 μl of the supernatant was collected as input control. The remaining extract was incubated with 25 µl anti-FLAG M2 affinity gel (Sigma-Aldrich, A2220) at 4 °C for 3 h. After 5 washing steps in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 % [v/v] glycerol, the immunoprecipitated proteins and input controls were subjected to SDS-PAGE followed by western blot. Blots were developed with anti-Flag-HRP (1:5000, Sigma-Aldrich, A8592) and anti-GFP (1:10000, Invitrogen) antibodies. Yeast complementation assay . Expression constructs for SlLHA1 or SlLHA4 under control of the yeast PMA1 promotor in pMP1745, and the SlPLL2 variants in pMP1612 were co-transformed into Saccharomyces cerevisiae strain RS-72  ( M ATα ; ade1 1 -100 , his4-519 , L eu2-3 , 1 12 , pPMA1:GAL1 ) using the LiAc method 25 . The empty vectors were used as negative controls. Transformed cells were grown on galactose medium (SG + His, pH 5.5). Galactose induces the expression of the yeast proton pump PMA1 from the GAL1 promoter. After transfer to glucose medium (SD + His, pH 5.5), growth depends on the activity of the plasmid-borne tomato proton pumps LHA1 and LHA4. To assess the effect of the PLL2 variants on tomato proton pump activity, three single yeast colonies from one transformation were diluted in liquid glucose medium to OD 600 =0.1 and 0.01. Five µl of each dilution were spotted on selective media using a multi-channel pipette. Plates were incubated at 30 °C for 3-5 days. Pictures of galactose plates were taken earlier (after 2 days) than those of glucose plates (after 3 days). The experiment was repeated three times with cells from independent transformations. Isolation of the yeast microsomal fraction. A single colony was grown in 10 ml liquid galactose medium (SG + His, pH 5.5) and incubated overnight in an orbital shaker at 200 rpm, 30 °C. The next day, it was transferred to 250 ml galactose medium and incubated overnight as before. The third day, this yeast culture was pelleted at room temperature, 800 g for 10 min, then inoculated into 500 ml liquid glucose medium (SD + His, pH 5.5) for 20 h at 200 rpm, 30 °C. Finally, cells were pelleted at 5000 g and resuspended in 6 ml cold water. Six ml yeast cells were then lysed in 3 ml homogenisation buffer (1 Vol 0.5 M Tris, pH 7.5, 1/100 Vol 0.5 M EDTA, 1/500 Vol 0.5 M DTT), 30 µl PMSF and 30 µl pepstatin with 23 g glass beads by vortexing. Extracts were cleared by centrifugation for 15 min at 10000 g, 4 °C. Microsomal membranes were collected from the supernatant by ultracentrifugation at 50000 g, 4°C for 45 min. STED10 (10% sucrose, 0.1 M Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT) was used to fill and balance the tubes. Membranes were resuspended in GTED20 (23% glycerol, 0.1 M Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT). Protein concentration was determined using the Bradford method with γ-globulin as the reference. Western blot over-lay (Far western blot). Microsomal fractions (100 µg) were denatured in SDS-PAGE sample buffer at 50°C for 10 min and separated by 8 % [v/v] SDS-PAGE (40% [w/v] acrylamide 37:1, 1.5 M Tris-HCl, pH 8.85, 10% SDS, 4% [v/v] TEMED, 12% [v/v] APS) and blotted to nitrocellulose membranes. For the detection of LHA1 and LHA4, membranes were blocked with 5% [w/v] skim milk in wash buffer (25 mM Tris, 150 mM NaCl and 0.1% [v/v] Tween 20, pH 7.4). A polyclonal serum against AtAHA2 C-terminus (aa 851-949; 1:5000) and anti-rabbit IgG-HRP (1:10000; Millipore) were used as the first and secondary antibodies respectively. To assess the phosphorylation status of the penultimate threonine at the C-terminus of the proton pumps, membranes were blocked with 5% [w/v] skim milk in far-western buffer (20 mM MES, 130 mM NaCl, 10 mM MgSO 4 and 100 µM CaCl 2, pH 6.5) for 1h at room temperature. Then membranes were transferred into 10 ml far-western buffer containing 40 μg RGS-His epitope tagged 14-3-3 (GF14ø) protein 23 and 5 µM fusicoccin (dissolved in 96% ethanol, Santa Cruz Biotechnology) for 1h at room temperature. 14-3-3 protein binding was detected using a monoclonal anti-RGS-6x-His antibody (1:1000; Qiagen) followed by anti-mouse-HRP (1:10000; Millipore) as the primary and secondary antibodies, respectively. Insect feeding assays . Manduca sexta eggs were collected and kept in a climate chamber at 16 h/26°C and 8 h/24°C light-dark cycle until the larvae hatched. Two freshly-hatched neonates were placed on the 2 nd true leaf (from bottom to top) of 4-week-old tomato plants and enclosed in white organza bags. The developing larvae were moved onto younger leaves when the tissue was consumed. Larval mass was measured every two days until all leaves were consumed. Quantification of proteinase inbibitor (PI-II) activity. Proteinase inhibitor activity was assayed in tomato leaf extracts as soybean trypsin inhibitor (STI) equivalents using a radial diffusion assay as previously described 53 . Briefly, leaf disc samples (150 mg) were collected into 2 mL screw-cap tubes containing 0.6 g ZrO 2 beads (2.8-3.2 mm; Mühlmeier, Germany) and flash-frozen in liquid nitrogen. Samples were homogenised in 500 µl extraction buffer (50 g/l PVPP, 18.6 g/l Na 2 EDTA, 5 g/l Na-diethyldithiocarbamate-trihydrate, 2 g/l phenylthiourea in 0.1 M Tris-HCl, pH 7.6 for 150 µg of tissue) in a Fisherbrand™ Bead Mill 24 homogenizer at 4 m/s for 2 x 30 s. The extract was cleared by centrifugation (15 min, 16100 g, 4°C). The supernatant was filled into ø 0.4 mm-wells punched into 12 x 12 cm plates containing 25 ml of 1.8% [w/v] agar with 2 mg bovine trypsin (Sigma-Aldrich) in 0.1 M Tris-HCl, pH 7.6). After 18 hours at 4°C, 25 ml substrate solution (6 mg N -acetyl- DL -phenylalanine-naphthyl ester, 12 mg Fast Blue B salt in 0.1 M Tris/20% [v/v] dimethyl formamide) were added to stain for trypsin activity during 1 h at 37 °C. The diameter of the clear inhibition zones surrounding each well was used to calculate trypsin protease inhibitor activity using a standard curve with soybean trypsin inhibitor (Sigma-Aldrich) as the reference. 16 plants were analyzed per genotype in two technical replicates. Rhizosphere acidification and alkalinization assays. Five to six-day-old wild-type and pll2 tomato seedlings grown on ATS plates were transferred onto pH indicator plates containing 0.002 % [w/v] bromocresol purple (BCP) in water-ager (0.5 % [w/v] agar, pH 6.5 adjusted with KOH). Pictures were taken after 24 h of incubation. To monitor systemin-induced alkalinization of the rhizosphere, seedlings were transferred from ATS plates to indicator plates adjusted to pH 5.5 with acetic acid. They were then sprayed with 1 μM systemin or water as the control. Pictures were taken before and 30 minutes after application. Phylogenetic analysis . Phylogenetic trees of Solanum lycopersicum and Arabidopsis thaliana proteins in the P-type H + -ATPase and POLTERGEIST-LIKE families were generated using PhyloGenes v4.1( www.phylogenes.org ) 54 . SlLHA1 (Solyc03g113400), SlLHA4 (Solyc07g017780), and SlPLL2 (Solyc06g076100) are highlighted in red. Confocal microscopy. Subcellular localization of GFP-fusion proteins transiently expressed in  N. benthamiana was imaged on a Zeiss LSM900 confocal microscope. The excitation and emission wavelengths were 488 and 509 nm for GFP, and 506 and 751 nm for FM4-64, respectively. Bimolecular fluorescence complementation (BiFC) in  N. benthamiana was analyzed on a Zeiss LSM700 confocal microscope. Fluorescence signals were recorded for GFP (excitation 488 nm, emission 518 nm) and YFP (excitation 488 nm, emission 518 nm). Multiple leaves of at least two independent plants were analyzed. Figures were prepared using open source software Fiji 55 . Statistical analysis. Statistical tests in phosphoproteomics were performed using R packages (Limma, factoextra, hypeR). Details of other statistical analyses are indicated in the figure legends. Unpaired two tailed Student’s t -test and one-way ANOVA with Tukey’s multiple comparisons test were performed in GraphPad Prism 9.0 (GraphPad Software, Inc.).  For all box plots, boxes range from 25th to 75th percentiles with the splitting line at the median. Whiskers extend to the minimum and maximum values if lower than 1.5x interquartile range. Declarations Acknowledgements We thank Claudia Oecking (University of Tübingen) and Xu Wang (University of Hohenheim) for helpful discussions, and Georg Felix (University of Tübingen) and Xu Wang for the 35S::SYR1:GFP and 35S::SYR1 expression constructs, respectively. Technical assistance by Bianca Bukowski and Latisha Alleyne-Lafleur is gratefully acknowledged. Funding: The work was supported by the Deutsche Forschungsgemeinschaft [SFB 1101 to ASc and ASti (project D06)]. Code availability R Scripts for phosphoproteomic analysis and the linear regression plot of Fig. 2a are available at GitHub ( https://github.com/shibalili/systemin-project ). All bioinformatic tools used in this study are cited in the Methods section. Data availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 56 partner repository with the dataset identifier PXD054229 Source data are provided with this paper. Author contributions Conceptualization: ASc, ASti. Investigation: RL, FHA. Visualization: RL, ASc. Funding acquisition: ASc, ASti. Supervision ASti, ASte. Writiting – original draft: RL, ASc. Writing – review and editing: all authors Competing interests The authors declare no competing interests. Additional information Extended data is available for this paper at https://doi.org... Supplementary information: The online version contains supplementary material available at https://doi.org.... Correspondence and requests for materials should be addressed to [email protected] or [email protected] References Pearce, G., Strydom, D., Johnson, S. & Ryan, C. A. A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins. Science 253, 895–898 (1991). Schilmiller, A. L. & Howe, G. A. Systemic signaling in the wound response. Curr. Opin. Plant Biol. 8, 369–377 (2005). Wang, L. et al. The systemin receptor SYR1 enhances resistance of tomato against herbivorous insects. Nature Plants 4, 152–156 (2018). Beloshistov, R. E. et al. Phytaspase-mediated precursor processing and maturation of the wound hormone systemin. New Phytol. 218, 1167–1178 (2018). Gust, A. A., Pruitt, R. & Nürnberger, T. Sensing danger: key to activating plant immunity. Trends Plant Sci. 2, 779–791 (2017). Lanfermeijer, F. C., Staal, M., Malinowski, R., Stratmann, J. W. & Elzenga, J. T. M. Micro-electrode flux estimation confirms that the Solanum pimpinellifolium cu3 mutant still responds to systemin. Plant Physiol. 146, 129–139 (2008). Felix, G. & Boller, T. Systemin induces rapid ion fluxes and ethylene biosynthesis in Lycopersicon peruvianum cells. Plant J. 7, 381–389 (1995). Moyen, C., Hammond-Kosack, K. E., Jones, J., Knight, M. R. & Johannes, E. Systemin triggers an increase of cytoplasmic calcium in tomato mesophyll cells: Ca 2+ mobilization from intra- and extracellular compartments. Plant Cell Environ 21, 1101–1111 (1998). Boller, T. Chemoperception of microbial signals in plant cells. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 189–214 (1995). Escocard de Azevedo Manhães, A. M., Ortiz-Morea, F. A., He, P. & Shan, L. Plant plasma membrane-resident receptors: Surveillance for infections and coordination for growth and development. J. Int. Plant Biol. 63, 79–101 (2021). Zipfel, C. et al. Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125, 749–760 (2006). Kunze, G. et al. The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16, 3496–3507 (2004). Wasternack, C. Jasmonates: An update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann. Bot. 100, 681–697 (2007). Haj Ahmad, F., Wu, X., Stintzi, A., Schaller, A. & Schulze, W. X. The systemin signaling cascade as derived from time course analyses of the systemin-responsive phosphoproteome. Mol. Cell. Proteomics 18, 1526–1542 (2019). Sobol, G., Chakraborty, J., Martin, G. B. & Sessa, G. The emerging role of PP2C phosphatases in tomato immunity. Mol Plant Microbe In 35, 737–747 (2022). Schaller, A. & Oecking, C. Modulation of plasma membrane H + -ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell 11, 263–272 (1999). Kumari, A., Chételat, A., Nguyen, C. T. & Farmer, E. E. Arabidopsis H + -ATPase AHA1 controls slow wave potential duration and wound-response jasmonate pathway activation. Proc. Natl. Acad. Sci USA 116, 20226–20231 (2019). Julien, J. L. & Frachisse, J. M. Involvement of the proton pump and proton conductance change in the wave of depolarization induced by wounding in Bidens pilosa . Can. J. Bot. 70, 1451–1458 (1992). Schaller, A. & Frasson, D. Induction of wound response gene expression in tomato leaves by ionophores. Planta 212, 431–435 (2001). Haruta, M., Gray, W. M. & Sussman, M. R. Regulation of the plasma membrane proton pump (H + -ATPase) by phosphorylation. Curr Opin Plant Biol 28, 68–75 (2015). Falhof, J., Pedersen, J. T., Fuglsang, A. T. & Palmgren, M. Plasma membrane H + -ATPase regulation in the center of plant physiology. Mol. Plant 9, 323–337 (2016). Svennelid, F. et al. Phosphorylation of Thr-948 at the C terminus of the plasma membrane H + -ATPase creates a binding site for the regulatory 14-3-3 protein. Plant Cell 11, 2379–2391 (1999). Jahn, T. et al. The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H + -ATPase. Plant Cell 9, 1805–1814 (1997). Eisenhaber, F. et al. Prediction of lipid posttranslational modifications and localization signals from protein sequences: big-Pi, NMT and PTS1. Nucleic Acids Res 31, 3631–3634 (2003). Fuglsang, A. T. et al. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+-ATPase by preventing interaction with 14-3-3 protein. Plant Cell 19, 1617–1634 (2007). Barbez, E., Dünser, K., Gaidora, A., Lendl, T. & Busch, W. Auxin steers root cell expansion via apoplastic pH regulation in Arabidopsis thaliana . Proc. Natl. Acad. Sci. U S A 114, E4884-E4893 (2017). DeFalco, T. A. et al. A conserved module regulates receptor kinase signalling in immunity and development. Nature Plants (2022). Park, C.-J. et al. Rice XB15, a protein phosphatase 2C, negatively regulates cell death and XA21-mediated innate immunity. PLOS Biology 6, e231 (2008). Spartz, A. K. et al. SAUR Inhibition of PP2C-D phosphatases activates plasma membrane H + -ATPases to promote cell expansion in Arabidopsis. Plant Cell 26, 2129–2142 (2014). Ren, H., Park, M. Y., Spartz, A. K., Wong, J. H. & Gray, W. M. A subset of plasma membrane-localized PP2C.D phosphatases negatively regulate SAUR-mediated cell expansion in Arabidopsis. PLoS genetics 14, e1007455 (2018). Lin, W. et al. TMK-based cell-surface auxin signalling activates cell-wall acidification. Nature 599, 278–282 (2021). Nuhse, T. S., Bottrill, A. R., Jones, A. M. E. & Peck, S. C. Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant J. 51, 931–940 (2007). Haruta, M., Sabat, G., Stecker, K., Minkoff, B. B. & Sussman, M. R. A peptide hormone and its receptor protein kinase regulate plant cell expansion. Science 343, 408–411 (2014). Wang, X., Li, R., Stintzi, A. & Schaller, A. Automated real-time monitoring of extracellular pH to assess early plant defense signaling. in Plant Peptide Hormones and Growth Factors Vol. 2731 Meth. Mol. Biol. (ed A. Schaller) 169–178 (Humana, 2024). Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 13, 2513–2526 (2014). R.CoreTeam. R: A language and environment for statistical computing.. R Foundation for Statistical Computing (2022). Wickham, H. & al., e. Welcome to the Tidyverse. J Open Source Softw 4, 1668 (2019). Wickham, H. ggplot2: Elegant graphics for data analysis . (Springer-Verlag New York, 2016). Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43, 20 (2015). Stekhoven, D. J. & Bühlmann, P. MissForest—non-parametric missing value imputation for mixed-type data. Bioinformatics 28, 112–118 (2011). Tibshirani, R., Walther, G. & Hastie, T. Estimating the number of clusters in a data set via the gap statistic. Journal of the Royal Statistical Society Series B: Statistical Methodology 63, 411–423 (2002). Kassambara, A. & Mundt, F. factoextra: Extract and visualize the results of multivariate data analyses (R package version 1.0.7). (2020). Schwacke, R. et al. MapMan4: A refined protein classification and annotation framework applicable to multi-omics data analysis. Mol. Plant 12, 879–892 (2019). TheTomatoGenomeConsortium. The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485, 635–641 (2012). Federico, A. & Monti, S. hypeR: an R package for geneset enrichment workflows. Bioinformatics 36, 1307–1308 (2019). Gleave, A. P. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203–1207 (1992). Wang, X., Robles Luna, G., Arighi, C. N. & Lee, J.-Y. An evolutionarily conserved motif is required for Plasmodesmata-located protein 5 to regulate cell-to-cell movement. Commun Biol 3, 291 (2020). Xing, H.-L. et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology 14, 327 (2014). Bosch, M. et al. Jasmonic acid and its precursor 12-oxophytodienoic acid control different aspects of constitutive and induced herbivore defenses in tomato. Plant Physiol. 166, 396–410 (2014). Cedzich, A. et al. The protease-associated (PA) domain and C-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase 3 (SlSBT3). J. Biol. Chem. 284, 14068–14078 (2009). Reichardt, S., Stintzi, A. & Schaller, A. Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates. bio-protocol 13, e4608 (2023). Li, R., Schaller, A. & Stintzi, A. Quantitative measurement of pattern-triggered ROS burst as an early immune response in tomato. in Plant Peptide Hormones and Growth Factors Vol. 2731 Meth. Mol. Biol. (ed A. Schaller) 157–167 (Humana, 2024). Bandoly, M., Grichnik, R., Hilker, M. & Steppuhn, A. Priming of anti-herbivore defence in Nicotiana attenuata by insect oviposition: herbivore-specific effects. Plant, Cell & Environment 39, 848–859 (2016). Zhang, P. et al. PhyloGenes: An online phylogenetics and functional genomics resource for plant gene function inference. Plant Direct 4, e00293 (2020). Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676–682 (2012). Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucl. Acids Res. 50, D543-D552 (2021). Additional Declarations There is NO Competing Interest. Supplementary Files supplementarymaterial.pdf Supplementary Table S1: Genotyping of CRISPR/Cas9 mutants Supplementary Table S2: PCR cloning primers Supplementary Table S3: Primers for genome editing and genotyping Supplementary Table S4: Primers for RT-qPCR ExtendedDataFig.docx Cite Share Download PDF Status: Published Journal Publication published 04 Jul, 2025 Read the published version in Nature Plants → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4919676","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Letter","associatedPublications":[],"authors":[{"id":359871083,"identity":"64a61d59-2062-4ebf-9e65-cf25759d2c31","order_by":0,"name":"Andreas Schaller","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-6872-9576","institution":"University of Hohenheim","correspondingAuthor":true,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Schaller","suffix":""},{"id":359871084,"identity":"b3bcefb0-827f-4561-830c-1386635b300f","order_by":1,"name":"Rong Li","email":"","orcid":"https://orcid.org/0000-0002-3098-611X","institution":"University of Hohenheim","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Li","suffix":""},{"id":359871085,"identity":"68a3e2f0-d6d4-40e6-a712-31e10f16e6d4","order_by":2,"name":"Fatima Haj Ahmad","email":"","orcid":"https://orcid.org/0000-0001-5347-3918","institution":"Al-Balqa Applied University","correspondingAuthor":false,"prefix":"","firstName":"Fatima","middleName":"Haj","lastName":"Ahmad","suffix":""},{"id":359871086,"identity":"9fa9ce39-ba71-4a3b-ba5e-5caa3b1d253d","order_by":3,"name":"Anja Fuglsang","email":"","orcid":"https://orcid.org/0000-0003-1153-8394","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Anja","middleName":"","lastName":"Fuglsang","suffix":""},{"id":359871087,"identity":"da8776fb-d5ef-4238-bca8-12f4641718de","order_by":4,"name":"Anke Steppuhn","email":"","orcid":"","institution":"University of Hohenheim","correspondingAuthor":false,"prefix":"","firstName":"Anke","middleName":"","lastName":"Steppuhn","suffix":""},{"id":359871088,"identity":"ff006c80-7222-476c-b4a1-82c16a40c532","order_by":5,"name":"Annick Stintzi","email":"","orcid":"https://orcid.org/0000-0002-6479-8184","institution":"University of Hohenheim","correspondingAuthor":false,"prefix":"","firstName":"Annick","middleName":"","lastName":"Stintzi","suffix":""}],"badges":[],"createdAt":"2024-08-15 13:47:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4919676/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4919676/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41477-025-02040-7","type":"published","date":"2025-07-04T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":67355219,"identity":"af1146b3-ada5-40ec-a326-3e7827b53d50","added_by":"auto","created_at":"2024-10-24 05:00:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":897652,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe systemin response is characterized by transient dephosphorylation of cellular proteins at 1 min after treatment. a\u003c/strong\u003e, Extracellular alkalinization is an early hall mark of phytocytokine signaling. The pH of the culture medium was recorded after addition 10 nM systemin or 20 nM flg22 to wild-type (WT) and \u003cem\u003esyr1\u003c/em\u003e cells. \u003cstrong\u003eb\u003c/strong\u003e, The systemin-induced phospho-proteomic response of WT cells is lost in \u003cem\u003esyr1\u003c/em\u003e. Principal Component Analysis (PCA) is shown for phospho-site data sets obtained in six biological replicates for each genotype and time point after systemin treatment. \u003cstrong\u003ec-e\u003c/strong\u003e, K-means clustering of phospho-sites (P-sites) according to their temporal changes in intensity identified five time profiles between the WT and \u003cem\u003esyr1\u003c/em\u003e cell culture. The black lines in (d) and (e) show the median of phospho-site intensity for each cluster . The number of sites in each cluster is indicated. A transient drop in intensity is frequently observed in WT but not in \u003cem\u003esyr1 \u003c/em\u003e(clusters 2 and 3 in (\u003cstrong\u003ec\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eand (\u003cstrong\u003ed\u003c/strong\u003e). \u003cstrong\u003ee\u003c/strong\u003e, Phosphosites that belong to clusters 2 and 3 in WT, distribute to other clusters in \u003cem\u003esyr1\u003c/em\u003e. \u003cstrong\u003ef\u003c/strong\u003e, Systemin-induced changes in phosphorylation at Ser151, Ser142 and Ser160 of SlPLL2. The profiles of these phospho-sites belong to cluster 2. For Ser151, which has data for each time point, medians are connected by a trend line.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4919676/v1/e09e842c88339d015e3b027a.png"},{"id":67356004,"identity":"3854e080-3ddd-4a90-b74a-30c15831a634","added_by":"auto","created_at":"2024-10-24 05:08:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":596134,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe dephosphorylated, active form of SlPLL2 inhibits tomato P-type H\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e-ATPases.\u003c/strong\u003e \u003cstrong\u003ea \u003c/strong\u003eand\u003cstrong\u003e b, \u003c/strong\u003eSlPLL2 activity depends on its phosphorylation status. SlPLL2\u003csup\u003eWT\u003c/sup\u003e, phospho-dead SlPLL2\u003csup\u003e3A\u003c/sup\u003e (where Ser\u003csup\u003e142-150-160\u003c/sup\u003e were mutated to Ala), phospho-mimetic SlPLL2\u003csup\u003e3D\u003c/sup\u003e (where Ser\u003csup\u003e142-150-160\u003c/sup\u003e were mutated to Asp) and free GFP  (negative control) were purified from agro-infiltrated \u003cem\u003eN. benthamiana \u003c/em\u003eleaves. Equal amounts of protein (Extended Data Fig. 3) were used with a synthetic phospho-peptide substrate (pLHA1, 100 µM) in \u003cem\u003ein-vitro\u003c/em\u003e assays based on the colorimetric quantification of phosphate release. \u003cstrong\u003ea\u003c/strong\u003e, Progress curves (linear regression) of phosphate release. \u003cstrong\u003eb\u003c/strong\u003e, Catalytic rates of SlPLL2 variants shown as pmol phosphate released per hour. Data represent the mean +/- SD of six independent experiments. Different letters are used to indicate statistically significant differences at \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05 (ANOVA followed by Tukey test). \u003cstrong\u003ec \u003c/strong\u003eand\u003cstrong\u003e d\u003c/strong\u003e, Co-IP assay of SlPLL2 interaction with LHA1 and LHA4. SlPLL2\u003csup\u003eWT\u003c/sup\u003e or its phospho-variants (SlPLL2\u003csup\u003e3A\u003c/sup\u003e, SlPLL2\u003csup\u003e3D\u003c/sup\u003e) were co-expressed as GFP fusions with Flag-tagged LHA4 (\u003cstrong\u003ec\u003c/strong\u003e) or LHA1 (\u003cstrong\u003ed\u003c/strong\u003e) in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Total protein extracts were immunoprecipitated with anti-Flag agarose beads, then detected with anti-GFP on western blots. CBB, Coomassie brilliant blue. All experiments were performed three times with similar results.\u003cstrong\u003e e, \u003c/strong\u003eSlPLL2\u003csup\u003eWT\u003c/sup\u003e interacts with LHA1 only after systemin-induced dephosphorylation. SlPLL2 phospho-variants were co-expressed as GFP fusions with Flag-tagged LHA1 in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Two minutes before harvesting, leaves were treated with 100 nM systemin or flg22 as indicated. The Co-IP assay was performed twice with similar results as described for c and d. \u003cstrong\u003ef\u003c/strong\u003e and \u003cstrong\u003eg\u003c/strong\u003e, Growth assays showing SlPLL2-mediated regulation of H\u003csup\u003e+\u003c/sup\u003e-ATPase activity in yeast. Yeast strain RS72 was (co-)-transformed with expressions constructs for tomato H\u003csup\u003e+\u003c/sup\u003e-ATPases LHA1 (\u003cstrong\u003eg\u003c/strong\u003e) or LHA4 (\u003cstrong\u003ef\u003c/strong\u003e) and SlPLL2 phospho-variants in different combinations. Growth on Gal medium is supported by the yeast endogenous proton pump (control). Growth on Glu medium depends on the activity of LHA1 and LHA4, which is modulated by co-expression of SlPLL2\u003csup\u003eWT\u003c/sup\u003e, SlPLL2\u003csup\u003e3A\u003c/sup\u003e and SlPLL2\u003csup\u003e3D\u003c/sup\u003e. For better comparison cells were spotted in two dilutions (OD\u003csub\u003e600\u003c/sub\u003e = 0.1 or 0.01) onto selective media at pH 5.5. The results are representative of three independent biological replicates. \u003cstrong\u003eh\u003c/strong\u003e and \u003cstrong\u003ei, \u003c/strong\u003ewestern blot overlay monitoring the phosphorylation status of the regulatory threonine of LHA1 and LHA4. Microsomal fractions (100 µg) of yeast cultures (co-)expressing LHA1 (\u003cstrong\u003ei\u003c/strong\u003e) or LHA4 (\u003cstrong\u003eh\u003c/strong\u003e) with different SlPLL2 variants were separated by SDS-PAGE and analyzed on western blots using an antiserum against the Arabidopsis H\u003csup\u003e+\u003c/sup\u003e-ATPase (AHA2). To monitor phosphorylation at the penultimate threonine, blots were incubated with recombinant RGS-His-tagged 14-3-3 protein and developed using a monoclonal antibody raised against the RGS-His tag. Experiments were performed three times with similar results.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4919676/v1/5bc7b012a05d9f457b9c6a53.png"},{"id":67356006,"identity":"c35eaf3d-6574-474d-a369-2d5c6047f93d","added_by":"auto","created_at":"2024-10-24 05:08:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":795484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSystemin responses depend on SlPLL2. a,\u003c/strong\u003e \u003cstrong\u003e\u003c/strong\u003e Loss of PLL2 function leads to increased proton extrusion in \u003cem\u003epll2\u003c/em\u003e compared to wild-type (WT) seedlings. Extracellular pH was monitored in the rhizosphere of 5-day-old seedlings transferred to pH indicator plates containing bromocresol purple (BCP) at pH 6.5. Changes in medium color were recorded after 24 h. \u003cstrong\u003ec,\u003c/strong\u003e Systemin-induced increase in extracellular pH depends on PLL2. 5-day-old seedlings were transferred to BCP indicator plates at pH 5.5. Medium color was recorded immediately (time 0) and 30 min after treatment with systemin (1 μM) or water (mock). \u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ed, \u003c/strong\u003eRoot growth is enhanced in \u003cem\u003epll2\u003c/em\u003e, and systemin-induced growth inhibition is impaired in \u003cem\u003epll2\u003c/em\u003e compared to WT seedlings\u003cstrong\u003e.\u003c/strong\u003e Root and hypocotyl length were measured for seedlings grown for 7 days on ATS medium. Statistically significant differences are indicated at *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003e P\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 and ns: not significant (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test). Box plots show n = 28-31 independent seedlings. \u003cstrong\u003ed,\u003c/strong\u003e Root and hypocotyl length of \u003cem\u003epll2\u003c/em\u003e and WT seedlings grown on ATS plates supplemented with 100 nM systemin are shown in percent of the average length reached on control plates. \u003cstrong\u003ee, \u003c/strong\u003eSystemin-induced ROS production is impaired in \u003cem\u003epll2\u003c/em\u003e.\u003cstrong\u003e \u003c/strong\u003eROS production was analyzed in leaf discs of 4-week-old tomato WT and \u003cem\u003epll2\u003c/em\u003e plants treated with 30 nM systemin. Progress curves show the mean +/- SE of 32 experimental plants for each genotype including three independent \u003cem\u003epll2 \u003c/em\u003ealleles. \u003cstrong\u003ef, I\u003c/strong\u003entensity of the systemin-induced ROS burst in SYR1-expressing \u003cem\u003eN. benthamiana\u003c/em\u003e plants correlates with PLL2 activity. The ROS burst was induced\u003cstrong\u003e \u003c/strong\u003eby addition of 30 nM systemin to leaf discs of \u003cem\u003eN. benthamiana\u003c/em\u003e plants co-expressing the SYR1 receptor and SlPLL2\u003csup\u003ewt\u003c/sup\u003e, SlPLL2\u003csup\u003e3A\u003c/sup\u003e or SlPLL2\u003csup\u003e3D\u003c/sup\u003e variants. Data represent the mean ± SE of n=39 independent plants. \u003cstrong\u003eg,\u003c/strong\u003e flg22-induced ROS burst in \u003cem\u003eN. benthamiana\u003c/em\u003e is unaffected by SlPLL2 activity. ROS production was analyzed in leaf discs of \u003cem\u003eN.\u003c/em\u003e \u003cem\u003ebenthamiana\u003c/em\u003e plants expressing SlPLL2-sfGFP variants after addition of 30 nM flg22. Data represent the mean ± SE of n=40 independent plants. \u003cstrong\u003eh\u003c/strong\u003e, Systemin-triggered MAPK activation is reduced in \u003cem\u003epll2\u003c/em\u003e. Leaf tissue was harvested from three-week-old plants in a time series after systemin (1 μM) treatment. Extracts (30 µg of protein) were analyzed by immunoblotting using an anti-pERK antibody. CBB: Coomassie Brilliant Blue. \u003cstrong\u003ei - k\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eInduction of \u003cem\u003ePI-II\u003c/em\u003e expression by systemin and wounding is impaired in \u003cem\u003epll2\u003c/em\u003e compared to WT plants. Three-week-old plants were treated with systemin (1 µM) or wounded by crushing with a hemostat. Leaf tissue was harvested before and 1, 3, and 6 hours after treatment for RNA extraction and RT-qPCR analysis. Relative \u003cem\u003ePI-II\u003c/em\u003e expression is shown as fold change over the untreated control. Results are shown as box plots with n=12 biological replicates. Asterisks indicate significant differences in induction levels between WT and \u003cem\u003epll2\u003c/em\u003e at *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003el - m\u003c/strong\u003e, Herbivore defense is impaired in \u003cem\u003epll2\u003c/em\u003e compared to WT plants. Freshly-hatched \u003cem\u003eM. sexta\u003c/em\u003e were placed on the 2\u003csup\u003end\u003c/sup\u003e oldest leaf of 4-week-old plants and, when all leaf material had been consumed, transferred to progressively younger leaves. \u003cstrong\u003el\u003c/strong\u003e, after five days, proteinase inhibitor (PI) activity was analyzed in systemic (unwounded) leaves. PI activity is shown as soybean trypsin inhibitor (STI) equivalents per gram fresh weight (FW). Data in box plots represent n = 16 plants. \u003cstrong\u003em\u003c/strong\u003e, Mass of \u003cem\u003eM. sexta\u003c/em\u003e larvae after two weeks of feeding. Data in box plots represent n = 37 larvae from 20 plants. The experiment was repeated twice with two independent \u003cem\u003epll2\u003c/em\u003e lines, showing similar results. \u003cstrong\u003el - m\u003c/strong\u003e, Statistically significant differences are indicated at *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 and ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4919676/v1/bc84b86a237831b239dccb90.png"},{"id":86057628,"identity":"5d46bcd1-67fd-49f6-a3ef-8840a45b83a6","added_by":"auto","created_at":"2025-07-05 07:10:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3447078,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4919676/v1/a7f04396-5dfa-44a5-aa34-6f6e26b81563.pdf"},{"id":67355222,"identity":"ac4f7d0e-2c10-47e8-af24-f27bee291156","added_by":"auto","created_at":"2024-10-24 05:00:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":245628,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table S1: Genotyping of CRISPR/Cas9 mutants\u003c/p\u003e\n\u003cp\u003eSupplementary Table S2: PCR cloning primers\u003c/p\u003e\n\u003cp\u003eSupplementary Table S3: Primers for genome editing and genotyping\u003c/p\u003e\n\u003cp\u003eSupplementary Table S4: Primers for RT-qPCR\u003c/p\u003e","description":"","filename":"supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4919676/v1/a2cfb757bf53c5fbc80f58f4.pdf"},{"id":67356005,"identity":"09c6a12f-6420-42ac-bcc2-914c08eb3ed8","added_by":"auto","created_at":"2024-10-24 05:08:34","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2388050,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-4919676/v1/a33056827c37d0203cf23a91.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Poltergeist-Like 2 (PLL2)-dependent activation of the wound response distinguishes systemin from other immune signaling pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLike other phytocytokines, the 18-amino-acid systemin peptide is derived from a larger precursor protein by proteolytic processing\u003csup\u003e4,5\u003c/sup\u003e. Rapid cellular responses to systemin include the extracellular alkalinization response, an increase in cytosolic calcium, depolarization of the plasma membrane, and the induction of an oxidative burst\u003csup\u003e3,6-8\u003c/sup\u003e. These early systemin responses resemble those triggered by other phytocytokines and are hallmarks of pattern-triggered immune signaling\u003csup\u003e9-12\u003c/sup\u003e. They depend on the systemin receptor SYR1 resembling many other peptide and pattern recognition receptors (PRRs) in the large family of leucine-rich receptor kinases (LRR-RKs)\u003csup\u003e3\u003c/sup\u003e. However, despite apparent similarities in perception mechanism and early signal transduction events, systemin induces distinct downstream responses including the activation of the octadecanoid pathway for the production of jasmonates that are responsible for the systemic induction of herbivore defense gene expression, rather than pattern-triggered immunity (PTI)\u003csup\u003e13\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo get insight into the specific signaling events downstream of the SYR1 receptor kinase, we used phospho-proteomics in a systemin-responsive tomato cell culture. In a previous study, we had compared the response of these cells to systemin and to an inactive control peptide\u003csup\u003e14\u003c/sup\u003e. Here, focusing on SYR1-dependent signaling events, we compared systemin-induced changes in protein phosphorylation in wild-type and \u003cem\u003esyr1\u0026nbsp;\u003c/em\u003emutant cell cultures, generated by biolistic transformation with a CRISPR/Cas9 genome editing construct (Supplementary Table S1). A rapid increase in medium pH, i.e. the systemin-induced alkalinization response, was observed for wild type but not for \u003cem\u003esyr1\u003c/em\u003e. Confirming the specific loss only of \u003cem\u003eSYR1\u0026nbsp;\u003c/em\u003efunction, the response to flg22 that depends on FLS2 as PRR was unaffected in \u003cem\u003esyr1\u003c/em\u003e cells (Fig. 1a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor phospho-proteomics, \u003cem\u003esyr1\u003c/em\u003e and wild-type cells were harvested in a time series after systemin treatment in six biological replicates (Extended Data Fig.1a, b). Microsomal membranes were isolated from each sample, digested with Lys-C and trypsin, enriched for phosphopeptides, and subjected to mass spectrometry (LC-MS/MS) in untargeted data-dependent acquisition mode. Quantitative information was obtained for 4804 phosphopeptide species matching 2155 different proteins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrincipal component (PC) analysis of phosphosites that responded to systemin treatment (sites differentially phosphorylated at\u0026nbsp;\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05, comparing all time points to time 0) clearly separated wild-type and \u003cem\u003esyr1\u003c/em\u003e samples. 43.4% of the total variance is explained by differences between the two genotypes (Fig. 1b). The second PC separated wild-type according to sampling time reflecting systemin-induced changes in protein phosphorylation. In contrast, all \u003cem\u003esyr1\u0026nbsp;\u003c/em\u003esamples clustered closely together indicating that there is no such systemin response in the receptor mutant (Fig. 1b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe then performed k-means clustering of phospho-peptide abundance profiles to compare dynamic changes in protein phosphorylation between the two cell cultures (Fig. 1d, Extended Data Fig. 2a). The largest difference was observed for clusters 2 and 3, characterized by a rapid and transient decrease in phosphorylation at 1 min after systemin treatment. Less than 20% (198) of the peptides in these clusters were from \u003cem\u003esyr1\u003c/em\u003e, compared to \u0026gt; 80% (1036) from the systemin-responsive wild type (Fig. 1c). We then checked whether these 1036 peptides show time-dependent changes in phosphorylation in \u003cem\u003esyr1\u003c/em\u003e cells and, if so, to which cluster they belong. We found them in all clusters except 2 and 3; none of them showed the systemin-induced drop in abundance at 1 min after treatment (Fig. 1e). The data indicate that the SYR1-mediated systemin response is characterized by rapid and transient de-phosphorylation of cellular proteins, implying the activation of protein phosphatases early in systemin signaling.\u003c/p\u003e\n\u003cp\u003eMost of the protein phosphatases showing SYR1-dependent changes in phosphorylation belonged to the PPM/PP2C family, and the largest number of phosphosites differentially regulated after systemin treatment was observed for poltergeist-like phosphatases (PLLs; Extended Data Fig. 2b,c), representing clade C in the PP2C family\u003csup\u003e15\u003c/sup\u003e. We focused our attention on PLL2 (Solyc06g076100) for which all three detected phosphosites showed a cluster 2-type phosphorylation response (Extended Data Fig. 2d). PLL2 was transiently dephosphorylated after systemin treatment at three sites in its N-terminal domain. For Ser151, phosphorylation was found to be decreased, for Ser142 and Ser160 it was reduced to below detection limits at 1 min after systemin treatment (Fig. 1f).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAddressing the effect of reversible phosphorylation on PLL2 activity, we performed site-directed mutagenesis to replace the three serines with phospho-mimetic aspartate, or non-phosphorylatable (phospho-dead) alanine residues. The wild-type enzyme (PLL2\u003csup\u003eWT\u003c/sup\u003e), as well as its phospho-mimic (PLL2\u003csup\u003e3D\u003c/sup\u003e) and phospho-dead (PLL2\u003csup\u003e3A\u003c/sup\u003e) variants were expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves as GFP fusions and purified by immuno-precipitation (GFP-trap; Extended Data Fig. 3). Phosphatase activity of the recombinant proteins was analyzed \u003cem\u003ein vitro\u003c/em\u003e as the amount of inorganic phosphate released from a synthetic phosphopeptide substrate. \u0026nbsp;PLL2\u003csup\u003e3A\u003c/sup\u003e showed three-fold higher phosphatase activity, while the phospho-mimic PLL2\u003csup\u003e3D\u003c/sup\u003e mutant was significantly less active compared to the wild-type enzyme (Fig. 2a, b). The data indicate that PLL2 is activated by dephosphorylation in response to systemin in a SYR1-dependent manner. Activated PLL2 may regulate downstream proteins by dephosphorylation, thereby contributing to the transient drop in phosphosite abundance observed after systemin treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMapMan was used to functionally characterize potential PLL2 targets among cluster 2 and 3 proteins showing the systemin-induced dephosphorylation response. Among the MapMan bins significantly enriched in clusters 2 and 3 are \u0026sbquo;solute carrier-mediated transport\u0026rsquo; (bin 24.2) and \u0026sbquo;solute primary active transport\u0026rsquo; (bin 24.1) (Extended Data Fig. 4a), including two plasma membrane proton pumps,\u0026nbsp;LHA1 (Solyc03g113400) and LHA4 (Solyc07g017780)\u0026nbsp;as putative\u0026nbsp;targets of PLL2 (Extended Data Fig. 4b). LHA1 was also suggested as a putative PLL target by correlation analysis of phosphorylation profiles in our previous study comparing phosphoproteomic responses to systemin and an inactive control peptide\u003csup\u003e14\u003c/sup\u003e. Here, we observed the dephosphorylation of LHA1 and of a second plasma membrane proton pump (LHA4) at 1 minute after systemin treatment. Transient dephosphorylation was more pronounced for LHA1 compared to LHA4 and it depended on SYR1 (Extended Data Fig. 4c, d), suggesting a role for LHA1 downstream of SYR1 in the systemin signaling pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPases have previously been implicated in systemin-signaling in tomato, and in wound signaling in general\u003csup\u003e16-18\u003c/sup\u003e. Reduced proton extrusion as a result of proton pump inhibition may contribute to extracellular alkalinization and membrane depolarization, that are both necessary and sufficient for the activation of downstream defense gene expression\u003csup\u003e16,19\u003c/sup\u003e. The activity of plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPases is controlled by reversible phosphorylation, with the second-to-last C-terminal threonine residue as the main regulatory site\u003csup\u003e20,21\u003c/sup\u003e. Phosphorylation of this residue and subsequent binding of 14-3-3 proteins stimulates proton pump activity, while dephosphorylation inactivates the enzyme\u003csup\u003e22,23\u003c/sup\u003e. This regulatory threonine is the site that was found to be dephosphorylated in LHA1 and LHA4 in response to systemin treatment (Extended Data Fig 4c, d). This is also the site that was dephosphorylated by PLL2 in the synthetic phospho-peptide we used as the substrate in our \u003cem\u003ein vitro\u003c/em\u003e activity assay (Fig. 2a, b). This assay showed that PLL2 dephosphorylates the regulatory phospho-threonine from the C-terminus of LHA1, with highest activity for the phospho-dead PLL2\u003csup\u003e3A\u003c/sup\u003e mutant (Fig. 2a, b). The data sugest that systemin-induced dephosphorylation and activation of PLL2 contribute to the alkalinization response by inhibition of plasma membrane proton pump activity. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo corroborate this hypothesis, we first tested whether PLL2 co-localizes with the proton pumps at the plasma membrane. A potential myristoylation site for plasma membrane targeting is tentatively predicted for PLL2\u003csup\u003e24\u003c/sup\u003e. \u0026nbsp;Plasma membrane localization was indeed observed for PLL2\u003csup\u003eWT\u003c/sup\u003e and also for the PLL2\u003csup\u003e3A\u0026nbsp;\u003c/sup\u003eand PLL2\u003csup\u003e3D\u003c/sup\u003e variants transiently expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves (Extended Data Fig. 5a). Interaction of PLL2 with the plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPases LHA1 and LHA4 was confirmed in\u003cem\u003e\u0026nbsp;N. benthamiana\u003c/em\u003e by bimolecular fluorescence complementation (BiFC; Extended Data Fig. 5b), and in co-immunoprecipitation (Co-IP) experiments testing for pull-down of GFP-tagged PLL2 by the flag-tagged regulatory (R) domain of the proton pumps. This experiment confirmed interaction of PLL2 with LHA4, which was observed for all three PLL2 variants (Fig. 2c). Hence, the interaction with LHA4 does not seem to depend on the phosphorylation status of PLL2. In contrast, only the hyperactive PLL2\u003csup\u003e3A\u003c/sup\u003e mutant was found to interact with the regulatory C-terminus of LHA1 (LHA1-R; Fig. 2d). For PLL2\u003csup\u003eWT\u003c/sup\u003e, interaction with LHA1-R was observed only after systemin treatment, which is consistent with the systemin-induced dephosphorylation and activation of PLL2 (Fig. 2e). No interaction was observed in response to flg22 (Fig. 2e). The data indicate that PLL2 needs to be in its dephosphorylated/activated state to interact with LHA1, and they further suggest that the SYR1-dependent dephosphorylation and activation of PLL2 distinguishes systemin from flg22-induced signaling.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo test whether PLL2 controls proton pump activity \u003cem\u003ein vivo\u003c/em\u003e, we first used the yeast strain RS-72 which expresses the endogenous H\u003csup\u003e+\u003c/sup\u003e-ATPase, \u003cem\u003ePMA1\u003c/em\u003e, under the control of the \u003cem\u003eGAL1\u003c/em\u003e promoter and, therefore, requires galactose as the sole carbon source for growth. On media containing glucose, the endogenous proton pump is not expressed and growth depends on the activity of plasmid-borne plant H\u003csup\u003e+\u003c/sup\u003e-ATPases\u003csup\u003e25\u003c/sup\u003e. Both LHA4 and LHA1 supported yeast growth indicating that the two pumps are expressed in yeast and active\u0026nbsp;(Fig. 2f, g). Binding of 14-3-3 proteins in a western blot overlay confirmed presence of the activating phospho-threonine at the regulatory C-termini of the proton pumps (Fig. 2h, i). Yeast growth and phosphorylation of LHA1 were suppressed upon co-expression of PLL2, and this inhibition depended on PLL2 activity: it was strongest for the hyperactive PLL2\u003csup\u003e3A\u0026nbsp;\u003c/sup\u003emutant, weaker for PLL2\u003csup\u003eWT\u003c/sup\u003e, and not observed for inactive PLL2\u003csup\u003e3D\u0026nbsp;\u003c/sup\u003e(Fig. 2g). \u0026nbsp;The data support our hypothesis that the alkalinization response depends on the systemin-mediated dephosphorylation and activation of PLL2 which, in turn, dephosphorylates and thereby inactivates the proton pump LHA1. PLL2-mediated inhibition of LHA4, on the other hand, did not seem to depend on the phosporylation status of PLL2. Yeast growth was impaired and phosphorylation of LHA4 was inhibited by co-expression of all three PLL2 variants (Fig. 2f, h). This result is consistent with our Co-IP experiments, which showed interaction of the regulatory LHA4 C-terminus with wild-type PLL2 as well as the PLL2\u003csup\u003e3A\u0026nbsp;\u003c/sup\u003eand PLL2\u003csup\u003e3D\u003c/sup\u003e mutants (Fig. 2c). Apparently, all three PLL2 variants are able to bind LHA4, irrespective of phosphorylation status, thereby preventing proton pump phosphorylation and activation, resulting in impaired yeast growth.\u003c/p\u003e\n\u003cp\u003eLoss-of-function analysis, using tomato \u003cem\u003epll2\u003c/em\u003e mutants generated by CRISPR/Cas9 genome editing (Extended Data Fig. 6) in root growth assays, corroborated PLL2-mediated regulation of proton pump activity and extracellular pH \u003cem\u003ein planta\u003c/em\u003e. As a readout for proton pump activity, we grew seedlings on pH indicator plates and monitored the acidification of the growth medium. Medium acidification was stronger for \u003cem\u003epll2\u003c/em\u003e than for wild-type seedlings (Fig. 3a). Consistent with enhanced proton pump activity and the \u0026lsquo;acid growth\u0026rsquo; hypothesis\u003csup\u003e26\u003c/sup\u003e, root growth was increased in \u003cem\u003epll2\u003c/em\u003e compared to wild type (Fig. 3b). The same assays were also used to test \u003cem\u003ein planta\u003c/em\u003e, whether PLL2 is required for systemin-mediated inhibition of proton pump activity and seedling growth. In wild-type seedlings, systemin treatment resulted in extracellular alkalinization, and this response was lost in \u003cem\u003epll2\u0026nbsp;\u003c/em\u003e(Fig. 3c). Consistent with increased cell wall pH, seedling growth was inhibited by systemin treatment in both roots and hypocotyls. Growth inhibition was substantially reduced in \u003cem\u003epll2\u003c/em\u003e compared to wild-type seedlings (Fig. 3d; Extended Data Fig. 7). The data confirm that systemin-induced pH responses depend on PLL2. The cumulative evidence supports our hypothesis that systemin signaling involves the SYR1-dependent activation of PLL2 which, in turn, dephosphorylates and thereby inactivates the proton pump LHA1 to result in the systemin-induced increase in extracellular pH and growth inhibition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe then tested whether PLL2 function is also required for downstream signaling and systemin-induced defense responses. The oxidative burst observed in wild-type plants in response to systemin treatment was clearly reduced in the \u003cem\u003epll2\u0026nbsp;\u003c/em\u003emutant (Fig. 3e). Reconstituting systemin signaling in \u003cem\u003eN. benthamiana\u003c/em\u003e, by co-expression of the SYR1 receptor with PLL2 or its phospho-mimic and phospho-dead mutants, we observed that the intensity of the systemin-induced ROS burst correlates with PLL2 activity. ROS production was highest for the hyperactive PLL2\u003csup\u003e3A\u0026nbsp;\u003c/sup\u003ecompared to PLL2\u003csup\u003eWT\u0026nbsp;\u003c/sup\u003eand the inactive PLL2\u003csup\u003e3D\u003c/sup\u003e mutant (Fig. 3f). Interestingly, the flg22-induced ROS burst was not affected by the tomato PLL2 variants suggesting differences in systemin and flg22-mediated regulation of ROS production (Fig. 3g). While the data do not preclude the possibility that PLL2 is involved in flg22 signaling as well, they indicate that the regulation of PLL2 activity by de-phosphorylation of serines 142, 151 and 160, is a specific feature of systemin signaling. In addition to the oxidative burst, the activation of mitogen-activated protein kinases (MAPKs), and the induction of defense gene (\u003cem\u003ePI-II\u003c/em\u003e) expression in response to systemin treatment or wounding were also compromised in \u003cem\u003epll2\u0026nbsp;\u003c/em\u003ecompared to wild type (Fig. 3h-k). As a result of impaired wound signaling, \u003cem\u003epll2\u003c/em\u003e accumulated lower amounts of defensive proteinase inhibitors than wild-type plants when fed upon by larvae of the specialist herbivore \u003cem\u003eManduca sexta\u0026nbsp;\u003c/em\u003e(Fig. 3l). Consequently, larvae gained weight more rapidly on \u003cem\u003epll2\u0026nbsp;\u003c/em\u003emutants compared to wild type (Fig. 3m) indicating a loss of insect resistance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we identified PLL2 as an element of the SYR1-dependent wound signaling pathway for induced herbivore defense. SYR1-dependent activation of PLL2 by dephosphorylation at three regulatory sites is required for the systemin-induced alkalinization response, ROS burst and MAPK activation. These early cellular systemin responses are also hallmarks of PTI. Interestingly and in contrast to tomato PLL2, close homologs in Arabidopsis (AtPLL4 and AtPLL5) and rice (XB15) have previously been identified as negative regulators of PTI\u003csup\u003e27,28\u003c/sup\u003e. These PLL2 homologs interact with multiple PRRs to dampen PTI. Upon ligand perception, AtPLL4 and 5 are phosphorylated at some of the seven predicted phosphorylation sites in their regulatory N-termini (Extended Data Fig. 8) and dissociate from the receptor complex. Thereby inhibition is released and PTI activated\u003csup\u003e27\u003c/sup\u003e. We conclude that closely related PLLs act downstream of SYR1 as well as PRRs, but are differentially regulated after perception of the respective ligands. Dephosphorylation and activation of PLL2 is observed specifically in response to systemin and may explain the systemin-specific induction of herbivore resistance, while the activation of PTI involves the phosphorylation of AtPLL4/5 to release PRRs from AtPLL4/5-mediated inhibition. \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe further report that PLL2 affects the proton gradient across the plasma membrane and extracellular pH by dephosphorylation and inhibition of plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPases.\u0026nbsp;PLL2 targets the penultimate phospho-threonine residue in the regulatory C-terminal domain of plasma membrane proton pumps LHA1 and LHA4. The same site is also targeted by auxin signaling. However, in contrast to PLL2-mediated proton pump inhibition and extracellular alkalinization, auxin involves a different phosphatase (PP2C-D) and a pair of antagonistic kinases (TMK1 and TMK4) to increase phosphorylation of the regulatory threonine, thereby stimulating proton extrusion and acid growth\u003csup\u003e29-31\u003c/sup\u003e. \u0026nbsp; In addition to the penultimate phospho-threonine residue, there are multiple other phosphosites in the regulatory C-terminus regulating proton pump activity, including serine 899. This serine is phosphorylated for proton pump inhibition in response to RALF and flg22\u003csup\u003e32,33\u003c/sup\u003e. Therefore, consistent with the specific role of PLL2 in systemin signaling, the regulatory mechanisms for proton pump inhibition appear to be different for the wound response and PTI. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant material and growth conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e cv. UC82B) and \u003cem\u003eN. benthamiana\u003c/em\u003e plants were cultivated in growth cabinets at 26 \u0026deg;C, with 16 h photoperiod, 100 \u003cem\u003e\u0026micro;mol m\u003csup\u003e-2\u003c/sup\u003e\u003c/em\u003e s\u003csup\u003e-1\u003c/sup\u003e light intensity and 75% rel. humidity. For seedling growth assays, tomato seeds were sterilized in 2% [v/v] bleach with 3 drops of Tween 20 per 25 ml, and then washed in sterile ddH\u003csub\u003e2\u003c/sub\u003eO. For germination, seeds were placed on ATS medium\u003csup\u003e30\u003c/sup\u003e containing 1% [w/v] sucrose. After 1-2 days, seeds that had just germinated were transferred onto either ATS control plates, or ATS plates containing 100 nM systemin for another 5-7 days of growth under the same condition as for plants. The tomato (\u003cem\u003eSolanum peruvianum\u003c/em\u003e) cell suspension culture was maintained as described\u003csup\u003e14,34\u003c/sup\u003e. For the \u003cem\u003esyr1\u003c/em\u003e mutant cell culture, the medium was supplemented with 75 mg/l kanamycin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElicitor and wound\u003c/strong\u003e\u003cstrong\u003eing treatments\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSystemin (AVQSKPPSKRDPPKMQTD) and the SlLHA1 C-terminal Thr955 \u0026nbsp;phosphopeptide (GLDIETIQQSYT (ph)V) were obtained from PepMic (Suzhou, China) at \u0026gt;95 % purity. Flg22 (QRLSTGSRINSAKDDAAGLQIA) was ordered at \u0026gt;95% purity from GenScript (USA). All peptides were dissolved in ddH\u003csub\u003e2\u003c/sub\u003eO at 1 mM and stored at -20 \u0026deg;C. For\u0026nbsp;mechanical wounding,\u0026nbsp;the first\u0026nbsp;two\u0026nbsp;true\u0026nbsp;leaves of 3-week-old tomato plants were\u0026nbsp;symmetrically\u0026nbsp;squeezed with a\u0026nbsp;hemostat\u0026nbsp;from both sides of the midrib\u0026nbsp;at each leaflet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhosphoproteomics and data\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003epreprocessing\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe setup of phosphoproteomics was adapted from\u0026nbsp;Haj Ahmad et al.\u003csup\u003e14\u003c/sup\u003e.\u0026nbsp;200 ml cell culture (wild-type and \u003cem\u003esyr1\u003c/em\u003e) was\u0026nbsp;harvested at\u0026nbsp;0, 1, 2, 5, 15, and 45 min after addition of 10 nM systemin.\u0026nbsp;Samples\u0026nbsp;were collected\u0026nbsp;from six independent batches of cells. Extraction of the microsomal fraction,\u0026nbsp;phosphopeptide enrichment and LC-MS/MS were conducted as previously described\u003csup\u003e14\u003c/sup\u003e.\u0026nbsp;Phosphosites were mapped against \u003cem\u003eSolanum lycopersicum\u003c/em\u003e ITAG3.2 by MaxQuant version 2.4.2.0\u003csup\u003e35\u003c/sup\u003e. Overall 4804 phosphosites were obtained excluding hits to\u0026nbsp;reverse sequences and potential contaminants, and\u0026nbsp;sites with localization probability \u0026le; 0.75. Phosphosite intensities\u0026nbsp;in the MaxQuant output table (Phospho (STY)Sites.txt) were\u0026nbsp;normalized\u0026nbsp;and log2\u0026nbsp;transformed\u0026nbsp;in R\u003csup\u003e36\u003c/sup\u003e version 4.1.0 (all R scripts are available at GitHub (\u003ca href=\"https://github.com/shibalili\"\u003ehttps://github.com/shibalili\u003c/a\u003e) under \u0026lsquo;systemin-project\u0026rsquo;).\u0026nbsp;Data manipulation and visualization were done using tidyverse\u003csup\u003e37\u003c/sup\u003e and ggplot2\u003csup\u003e38\u003c/sup\u003e packages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExploratory data analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe R package Limma\u003csup\u003e39\u003c/sup\u003e was used to analyze differentially phosphorylated phosphosites. For each phosphosite, a linear model was fit to describe its phosphorylation profile over time. Coefficients for each time point were compared to time 0 in multiple pairwise comparison.\u0026nbsp;Sites\u0026nbsp;differentially\u0026nbsp;phosphorylated\u0026nbsp;relative to the same genotype\u0026ndash;treatment combination at time 0 were identified at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u0026nbsp;Phosphosites which were\u0026nbsp;differentially\u0026nbsp;phosphorylated (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05) in WT cell culture were considered for\u0026nbsp;Principal Component Analysis (PCA)\u0026nbsp;using the prcomp function in R.\u0026nbsp;For analyses that require a complete set of data points for each phosphosite (e.g. k means clustering), missing values were imputed using\u0026nbsp;the R package missForest\u003csup\u003e40\u003c/sup\u003e. A relatively loose\u0026nbsp;cut-off of \u0026gt; 10 of maximum 72 data points per phosphosite was applied, which allowed 2450 (50 %) of the phosphosites with variable phosphorylation patterns to be retained for cluster analysis. Gap statistics\u003csup\u003e41\u003c/sup\u003e was applied to identify the optimal number of clusters as 5 (Extended Data Fig. 1b). Temporal phosphorylation profiles were clustered\u0026nbsp;using\u0026nbsp;the\u0026nbsp;eclust\u0026nbsp;function together with\u0026nbsp;kmeans and euclidean\u0026nbsp;distance from the R package factoextra\u003csup\u003e42\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMapMan\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;enrichment analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunctional annotation of proteins was done using\u0026nbsp;MapMan\u003csup\u003e43\u003c/sup\u003e ontology downloaded from MapManstore (\u003ca href=\"https://mapman.gabipd.org/mapmanstore\"\u003ehttps://mapman.gabipd.org/mapmanstore\u003c/a\u003e), and the\u0026nbsp;\u003cem\u003eSolanum lycopersicum\u003c/em\u003e genome annotation ITAG3.2\u003csup\u003e44\u003c/sup\u003e from\u0026nbsp;PHYTOZOME V13.0 (https://phytozome-next.jgi. doe.gov/).\u0026nbsp;Enrichment analysis was performed with the package hypeR\u003csup\u003e45\u003c/sup\u003e, using hypergeometric enrichment test to determine if a group of proteins is over-represented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Cloning.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePolymerase chain reaction (PCR) primers for\u0026nbsp;DNA\u0026nbsp;amplification from plant genomic DNA or plasmid templates are listed in Supplementary Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo generate expression clones for full-length wild-type\u003cem\u003e\u0026nbsp;SlPLL2\u003c/em\u003e (\u003cem\u003eSlPLL2\u003csup\u003eWT\u003c/sup\u003e\u003c/em\u003e), a synthetic DNA fragment (Integrated DNA Technology, IDT) corresponding to the first 936 bp of PLL2 was inserted\u0026nbsp;into pDONR221 by BP reaction (Invitrogen). The catalytic domain\u003csup\u003e14\u003c/sup\u003e was added by restriction enzyme cloning. To generate the \u003cem\u003eSlPLL2\u003csup\u003e3A\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003eSlPLL2\u003csup\u003e3D\u003c/sup\u003e\u003c/em\u003e mutants, a \u003cem\u003eCla\u003c/em\u003eI(277)-\u003cem\u003eBcl\u003c/em\u003eI(509) fragment of \u003cem\u003eSlPLL2\u003csup\u003eWT\u003c/sup\u003e\u003c/em\u003e including serines 142, 151 and 160 was replaced with synthetic fragments in which the respective codons had been replaced by GCA for alanine, or GAT for aspartate, respectively.\u0026nbsp;\u0026nbsp;To generate \u003cem\u003eSlPLL2\u003csup\u003eWT/3A/3D\u003c/sup\u003e-sfGFP\u003c/em\u003e constructs for transient expression in \u003cem\u003eN. benthamiana\u003c/em\u003e, the \u003cem\u003eSlPLL2\u003csup\u003eWT/3A/3D\u003c/sup\u003e\u003c/em\u003e variants and the coding sequence of sfGFP were amplified by PCR and inserted into pART7 between the CaMV 35S promoter and terminator. After \u003cem\u003eNot\u003c/em\u003eI digestion, this expression cassette was transferred into\u0026nbsp;pART27\u003csup\u003e46\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the BiFC assay, the following fusion constructs were generated:\u0026nbsp;SlLHA1-YFP, SlLHA1-nYFP, SlLHA4-YFP, SlLHA4-nYFP and SlPLL2-cYFP. To this end, the open reading frames (ORFs) of SlLHA1 (Solyc03g113400) and SlLHA4 (Solyc07g017780) were\u0026nbsp;PCR-amplified from tomato root cDNA and introduced into the\u0026nbsp;\u003cem\u003eXho\u003c/em\u003eI and \u003cem\u003eCla\u003c/em\u003eI sites\u0026nbsp;of pART7. The ORFs for full-length EYFP (residues 1-240) and the N-terminal fragment of EYFP (nYFP, residues 1-155) were inserted as\u0026nbsp;the \u003cem\u003eCla\u003c/em\u003eI-\u003cem\u003e\u0026nbsp;Xba\u003c/em\u003eI\u0026nbsp;fragment downstream of SlLHA1/4 to result in the \u003cem\u003eSlLHA1/4-YFP\u0026nbsp;\u003c/em\u003eand \u003cem\u003eSl\u003c/em\u003e\u003cem\u003eL\u003c/em\u003e\u003cem\u003eHA1/\u003c/em\u003e\u003cem\u003e4-nYFP\u003c/em\u003e fusions, respectively.Similarly, ORFs for SlPLL2 and the C-terminal fragment of EYFP (cYFP,\u0026nbsp;residues 156-240) were fused to result in\u0026nbsp;\u003cem\u003eSl\u003c/em\u003e\u003cem\u003ePLL2-cYFP\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the Co-IP assay, the following expression constructs were generated: \u003cem\u003eSYR\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e\u003cem\u003e, 3xFLAG-LHA1-R\u003c/em\u003e and \u003cem\u003e3xFLAG-\u003c/em\u003e\u003cem\u003eL\u003c/em\u003e\u003cem\u003eHA\u003c/em\u003e\u003cem\u003e4\u003c/em\u003e\u003cem\u003e-R\u003c/em\u003e,\u0026nbsp;in addition to\u003cem\u003e\u0026nbsp;Sl\u003c/em\u003e\u003cem\u003ePLL2\u003csup\u003eWT\u003c/sup\u003e-sfGFP\u003c/em\u003edescribed above.\u0026nbsp;A genomicfragment comprising \u003cem\u003eSYR1\u003c/em\u003e (Solyc03g082470) was PCR-amplified from genomic DNA and cloned into\u0026nbsp;pMB35S\u003csup\u003e47\u003c/sup\u003e using the \u003cem\u003eEco\u003c/em\u003eRV and \u003cem\u003eXba\u003c/em\u003eI sites.\u0026nbsp;The regulatory domain of\u0026nbsp;SlLHA1 (aa 864-956) and\u0026nbsp;SlLHA4\u0026nbsp;(aa 859-952) were PCR-amplified from full-length cDNA templates. The 3xFLAG tag sequence was included in the PCR primers and added at the N-terminus of\u0026nbsp;SlLHA1 and\u0026nbsp;SlLHA4.\u003c/p\u003e\n\u003cp\u003eThe yeast complementation assay required expression vectors for \u003cem\u003eSl\u003c/em\u003e\u003cem\u003eL\u003c/em\u003e\u003cem\u003eHA\u003c/em\u003e\u003cem\u003e4\u003c/em\u003e and \u003cem\u003eSlLHA1\u003c/em\u003e under control of the endogenous \u003cem\u003ePMA1\u0026nbsp;\u003c/em\u003epromoter. For this purpose, the \u003cem\u003eAtAHA2\u003c/em\u003e ORF in the previously described \u003cem\u003epPMA1:AtAHA2\u003c/em\u003e expression plasmid pMP1745 was replaced by \u003cem\u003eSl\u003c/em\u003e\u003cem\u003eL\u003c/em\u003e\u003cem\u003eHA\u003c/em\u003e\u003cem\u003e4\u003c/em\u003e or \u003cem\u003eSlLHA1\u0026nbsp;\u003c/em\u003eby \u003cem\u003eXho\u003c/em\u003eI-\u003cem\u003eNde\u003c/em\u003eI digestion\u003csup\u003e25\u003c/sup\u003e.\u0026nbsp;\u003cem\u003eSl\u003c/em\u003e\u003cem\u003ePLL2\u003c/em\u003e and its phosphor-variants were cloned into the pMP1612 expression vector using \u003cem\u003eNot\u003c/em\u003eI\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor CRISPR/Cas9-mediated genome editing, single guide RNA (sgRNA) constructs were generated in the pKSE401 binary vector comprising the pCaMV35S::Cas9 expression construct\u003csup\u003e48\u003c/sup\u003e.\u0026nbsp;CRISPRdirect (\u003ca href=\"https://crispr.dbcls.jp\"\u003ehttps://crispr.dbcls.jp\u003c/a\u003e) was used to select guide RNAs that included a restriction site at the location of the double strand break to facilitate subsequent genotyping. sgRNA expression cassettes were generated by PCR using primers containing the sgRNA sequence and a \u003cem\u003eBsa\u003c/em\u003eI site for golden gate cloning, and pCBCT1T2 and/or pCBCT2T3 as the template. Primer sequences are listed in Supplementary Table 3. The resulting pKSE401-gSYR1 and pKSE401-gPLL2 editing constructs contained 2 and 3 sgRNAs for the genomic \u003cem\u003eSlSYR1\u003c/em\u003e and \u003cem\u003eSlPLL2\u003c/em\u003e loci, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant transformation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCRISPR/Cas9 vectors were introduced into the cotyledons of 10-day-old etiolated tomato seedlings via \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e (GV3101)-mediated transformation. Transgenic lines were selected on media with kanamycin concentrations increasing successively from 35 mg/l to100 mg/l as described\u003csup\u003e49\u003c/sup\u003e.\u0026nbsp;The \u003cem\u003esyr1\u0026nbsp;\u003c/em\u003emutant cell culture was also generated by CRISPR/Cas9 genome editing. The editing construct was linearized with \u003cem\u003ePme\u003c/em\u003eI and shot into\u0026nbsp;\u003cem\u003eS\u003c/em\u003e\u003cem\u003e. peruvianum\u0026nbsp;\u003c/em\u003ecells using the PDS1000/He Biolistic Particle Delivery System (BioRad) as described\u003csup\u003e50\u003c/sup\u003e.\u0026nbsp;Transformed cells were selected on 75 mg/ml kanamycin for callus growth. Suspension cultures were established from calli that were confirmed by PCR to carry the editing construct and genotyped as follows.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping of CRISPR/Cas9 mutants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA was isolated from WT tomato plants or \u003cem\u003eS. peruvianum\u003c/em\u003e calli and corresponding CRISPR/Cas9 transformants. For transgenic plants, three independent \u003cem\u003esyr1\u003c/em\u003e and\u003cem\u003eslpll2\u003c/em\u003e mutants were identified by PCR and sequenced in the T0 generation (primers for genotyping are listed in Supplementary Table S3).\u0026nbsp;Homozygous mutants from the segregating\u0026nbsp;Cas9-free T2 progeny were used for experiments (Supplementary Table S1). For transgenic cell cultures generated by particle bombardment, genotyping was done by cloning the PCR-amplified target loci in pCR2.1-TOPO (Thermo Fisher Scientific). Eight clones per cell culture were sequenced revealing different mutations at the target site of the sgRNA and confirming absence of the wild-type sequence (Supplementary Table S1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA isolation and Reverse Transcriptase (RT)-qPCR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from leaves of 3-week-old tomato plants with\u0026nbsp;TRIzol reagent (Invitrogen) according to the manufacturer\u0026rsquo;s instructions. The RNA concentration was measured at 260 nm using a microplate reader (Tecan Spark) after DNase I digestion (Thermo Scientific).\u0026nbsp;One \u0026mu;g RNA was reversely transcribed to synthesize the cDNA with RevertAid reverse transcriptase (Thermo Fisher Scientific) and oligo(dT) primers. Real-time PCR (qPCR) was performed in a Bio-Rad CFX Connect real-time PCR instrument (Bio-Rad; Munich, Germany) with SYBR-Green (Cambrex Bio Science Rockland Inc.; Rockland, ME, USA). RT-qPCR primers are listed in Supplementary Table S4. Target gene expression was\u0026nbsp;analyzed\u0026nbsp;by delta-delta Ct method\u0026nbsp;and\u0026nbsp;normalized to the expression level of three house-keeping genes, \u003cem\u003eUBQ10\u003c/em\u003e, \u003cem\u003eACTIN2\u003c/em\u003e and\u0026nbsp;\u003cem\u003eEF1\u003c/em\u003e\u003cem\u003e\u0026alpha;\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransient protein expression in \u003cem\u003eN. benthamiana\u003c/em\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransient expression experiments were performed essentially as described\u003csup\u003e51\u003c/sup\u003e.\u0026nbsp;Briefly, agrobacteria carrying binary vectors with expression constructs for the protein(s) of interest and the P19 silencing suppressor were mixed to result in OD\u003csub\u003e600\u003c/sub\u003e = 0.5.\u0026nbsp;When PLL2 mutants were expressed for activity assays, the\u0026nbsp;OD\u003csub\u003e600\u003c/sub\u003e ratio (PLL2\u003csup\u003eWT/3A/3D\u003c/sup\u003e or free GFP : P19)\u0026nbsp;was\u0026nbsp;9:1.\u0026nbsp;\u003cem\u003eN. benthamiana\u003c/em\u003e lacks a functional systemin perception system. Therefore, in order to reconstitute systemin signaling in \u003cem\u003eN. benthamiana\u003c/em\u003e, a SYR1-eGFP expression construct kindly provided by Georg Felix (University of T\u0026uuml;bingen) was always co-infiltrated with the\u0026nbsp;PLL2 constructs (PLL2\u003csup\u003eWT/3A/3D\u0026nbsp;\u003c/sup\u003e: SYR1 : P19 = 8:1:1).\u0026nbsp;For Co-immunoprecipitation, the OD\u003csub\u003e600\u003c/sub\u003e ratio was PLL2\u003csup\u003eWT/3A/3D\u0026nbsp;\u003c/sup\u003e: LHA1/4 : P19 = 4.5:4.5:1, or PLL2\u003csup\u003eWT\u0026nbsp;\u003c/sup\u003e: LHA1 : SYR1 : P19 = 4:4:1:1.The infiltration\u0026nbsp;buffer\u0026nbsp;contained 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM\u0026nbsp;MES,\u0026nbsp;pH 5.6 and 150\u0026nbsp;\u0026mu;M acetosyringone.\u0026nbsp;Leaves were harvested 3 days post infiltration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlkalinization\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;assay.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMedium\u0026nbsp;alkalinization\u0026nbsp;in response to systemin (10 nM), flg22 (20 nM) and ddH\u003csub\u003e2\u003c/sub\u003eO as the control was analyzed in \u003cem\u003eS. peruvianum\u0026nbsp;\u003c/em\u003ewild-type and \u003cem\u003esyr1\u0026nbsp;\u003c/em\u003ecell suspensions seven days after subculture as described previously\u003csup\u003e34\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eROS burst.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eROS production in response to systemin (30 nM), flg22 (30 nM) and ddH\u003csub\u003e2\u003c/sub\u003eO as the control was analyzed in 4 mm leaf discs of 4-week-old tomato or \u003cem\u003eN. benthamiana\u003c/em\u003e plants as described previously\u003csup\u003e52\u003c/sup\u003e\u003csup\u003e.\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAPK activation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree-week-old tomato plants with two true leaves were dipped\u0026nbsp;briefly\u0026nbsp;into 1\u0026nbsp;\u0026mu;M systemin in 0.05%\u0026nbsp;[v/v]\u0026nbsp;silwet-77 or the solvent alone, and at\u0026nbsp;the indicated time\u0026nbsp;points, leaf tissue was harvested.\u0026nbsp;100 mg tissue samples were ground\u0026nbsp;in 100\u0026nbsp;\u0026mu;l extraction buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.5% [v/v] Triton X-100, 10 mM \u0026szlig;-mercaptoethanol, 1:1000 Protease Inhibitor Mix P (SERVA), 1:2000 Phosphatase Inhibitor Cocktail 2 and 1:2000 Phosphatase Inhibitor Cocktail 3 (Sigma))\u0026nbsp;using a bead beater (TissueLyzer LT; QIAGEN). The supernatant was collected after centrifugation at\u0026nbsp;16100 g\u0026nbsp;for 10 min at 4 \u0026deg;C. Protein samples were mixed with SDS loading buffer, denatured at 95 \u0026deg;C for 5 min, separated by 10%\u0026nbsp;[w/v]\u0026nbsp;SDS\u0026ndash;PAGE, and blotted to a nitrocellulose membrane. Western blots were developed to detect phosphorylated MAPKs using anti-pERK1/2 (1:5000; Cell Signaling) as the primary and goat anti-rabbit IgG (1:10000; Millipore) as the secondary antibodies, followed by enhanced chemiluminescence detection in a LICORbio Odyssey XF imager.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;phosphatase assay.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pART27-based expression constructs harboring phosphatase (SlPLL2\u003csup\u003eWT\u003c/sup\u003e,\u0026nbsp;SlPLL2\u003csup\u003e3A\u003c/sup\u003e and\u0026nbsp;SlPLL2\u003csup\u003e3D\u003c/sup\u003e)-sfGFP were agro-infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Three days post-infiltration, 100 mg leaf samples were harvested and ground in 100\u0026nbsp;\u0026mu;l extraction buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.5% [v/v] Triton X-100, 10 mM \u0026szlig;-mercaptoethanol, 1:1000 Protease Inhibitor Mix P) using a bead beater (TissueLyser LT; Qiagen). The extracts were cleared by centrifugation. An aliquot of the supernatant was used for the quantification of phosphatase expression levels by anti-GFP Western Blot analysis. The remainder was subjected to GFP-trap\u0026nbsp;(Chromotek) to immuno-precipitate SlPLL2 variants according to the manufacturer\u0026rsquo;s instructions. On-bead phosphatase activity assays were performed using the Serine/Threonine Phosphatase Assay Kit (Promega), with the bead volume adjusted to result in equal amounts of enzyme for each of the PLL2 variants (Extended Data Fig. 3). The phospho-peptide GLDIETIQQSYT\u0026nbsp;(ph)V, custom-synthesized at \u0026gt;95 % purity (PepMic; Suzhou, China),\u0026nbsp;was used as the substrate\u0026nbsp;at 100 \u0026micro;M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConstructs bearing phosphatase-sfGFP fusion constructs and the 3xFLAG-tagged regulatory domains of LHA1 and\u0026nbsp;LHA4\u0026nbsp;were transformed in \u003cem\u003eA. tumefaciens\u003c/em\u003e and co-infiltrated in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves in the combinations indicated. For elicitor treatments, the leaves co-expressing phosphatases and proton pumps were infiltrated with 100 nM systemin\u0026nbsp;or\u0026nbsp;100 nM flg22, harvested and flash-frozen in liquid nitrogen within\u0026nbsp;2\u0026nbsp;min.\u0026nbsp;Two infiltrated leaves (~2 g) were\u0026nbsp;ground in 2 ml\u0026nbsp;cold\u0026nbsp;IP buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 % [v/v] glycerol, 5\u0026nbsp;\u0026mu;M DTT,\u0026nbsp;0.5%\u0026nbsp;[v/v]\u0026nbsp;IGEPAL,\u0026nbsp;1 mM\u0026nbsp;PMSF, 1:1000 Protease Inhibitor Mix P from SERVA) on ice. The extract was cleared by centrifugation (16100 g, 15 min, 4 \u0026deg;C) and 50\u0026nbsp;\u0026mu;l of the supernatant was collected as input control. The remaining extract was incubated with 25 \u0026micro;l anti-FLAG M2 affinity gel (Sigma-Aldrich, A2220) at 4 \u0026deg;C for 3 h. After 5 washing steps in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 % [v/v]\u0026nbsp;glycerol, the immunoprecipitated proteins and input controls were subjected to SDS-PAGE followed by western blot. Blots were developed with anti-Flag-HRP (1:5000, Sigma-Aldrich, A8592) and anti-GFP (1:10000, Invitrogen) antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast complementation assay\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression constructs for \u003cem\u003eSlLHA1\u003c/em\u003e or \u003cem\u003eSlLHA4\u003c/em\u003e under control of the yeast \u003cem\u003ePMA1\u0026nbsp;\u003c/em\u003epromotor in pMP1745, and the\u0026nbsp;\u003cem\u003eSlPLL2\u003c/em\u003e variants in pMP1612 were co-transformed into\u0026nbsp;\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strain RS-72\u0026nbsp;\u0026nbsp;(\u003cem\u003eM\u003c/em\u003e\u003cem\u003eAT\u0026alpha;\u003c/em\u003e; \u003cem\u003eade1\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e\u003cem\u003e-100\u003c/em\u003e,\u0026nbsp;\u003cem\u003ehis4-519\u003c/em\u003e,\u0026nbsp;\u003cem\u003eL\u003c/em\u003e\u003cem\u003eeu2-3\u003c/em\u003e,\u0026nbsp;\u003cem\u003e1\u003c/em\u003e\u003cem\u003e12\u003c/em\u003e,\u0026nbsp;\u003cem\u003epPMA1:GAL1\u003c/em\u003e)\u0026nbsp;using the LiAc method\u003csup\u003e25\u003c/sup\u003e. The empty vectors were used as negative controls. Transformed cells were grown on galactose medium (SG + His, pH 5.5). Galactose induces the expression of the yeast proton pump PMA1 from the \u003cem\u003eGAL1\u003c/em\u003e promoter. After transfer to glucose medium (SD + His, pH 5.5), growth depends on the activity of the plasmid-borne tomato proton pumps LHA1 and LHA4. To assess the effect of the PLL2 variants on tomato proton pump activity, three single yeast\u0026nbsp;colonies from one transformation\u0026nbsp;were\u0026nbsp;diluted in liquid glucose medium to OD\u003csub\u003e600\u003c/sub\u003e=0.1 and 0.01. \u0026nbsp;Five \u0026micro;l of each dilution\u0026nbsp;were spotted\u0026nbsp;on selective media using a multi-channel pipette. Plates were incubated at 30\u0026nbsp;\u0026deg;C for 3-5 days. Pictures of galactose\u0026nbsp;plates were\u0026nbsp;taken earlier (after\u0026nbsp;2 days) than those of glucose plates (after 3 days).\u0026nbsp;The experiment was repeated three times with cells from independent transformations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of the yeast microsomal fraction.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA single colony was grown in 10 ml liquid galactose medium (SG + His, pH 5.5) and incubated overnight in an orbital shaker at 200 rpm, 30 \u0026deg;C. The next day, it was transferred to 250 ml galactose medium and incubated overnight as before. The third day, this yeast culture was pelleted at room temperature, 800 g for 10 min, then inoculated into 500 ml liquid glucose medium (SD + His, pH 5.5) for 20 h at 200 rpm, 30 \u0026deg;C. Finally, cells were pelleted at 5000 g and resuspended in 6 ml cold water. Six ml yeast cells were then lysed in 3 ml homogenisation buffer (1 Vol 0.5 M Tris, pH 7.5, 1/100 Vol 0.5 M EDTA, 1/500 Vol 0.5 M DTT), 30 \u0026micro;l PMSF and 30 \u0026micro;l pepstatin with 23 g glass beads by\u0026nbsp;vortexing. Extracts were cleared by centrifugation for 15 min at 10000 g, 4 \u0026deg;C. Microsomal membranes were collected from the supernatant by ultracentrifugation at 50000 g,\u0026nbsp;4\u0026deg;C\u0026nbsp;for 45 min. STED10 (10% sucrose, 0.1 M Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT) was used to fill and balance the tubes. Membranes were\u0026nbsp;resuspended in GTED20 (23% glycerol, 0.1 M Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT). Protein concentration was determined using the Bradford method with \u0026gamma;-globulin as the reference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eover-lay (Far western blot).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicrosomal fractions (100 \u0026micro;g) were denatured in SDS-PAGE sample buffer at 50\u0026deg;C for 10 min and separated\u0026nbsp;by\u0026nbsp;8 %\u0026nbsp;[v/v]\u0026nbsp;SDS-PAGE (40% [w/v]\u0026nbsp;acrylamide 37:1, 1.5 M Tris-HCl, pH 8.85, 10% SDS, 4% [v/v]\u0026nbsp;TEMED, 12% [v/v] APS) and blotted to nitrocellulose membranes.\u0026nbsp;For the detection of LHA1 and LHA4, membranes were blocked with 5% [w/v] skim milk in wash buffer (25 mM Tris, 150 mM NaCl and 0.1% [v/v] Tween 20, pH 7.4). A polyclonal serum against AtAHA2 C-terminus (aa 851-949; 1:5000) and\u0026nbsp;anti-rabbit IgG-HRP\u0026nbsp;(1:10000; Millipore) were used as the first and secondary antibodies respectively. To assess the phosphorylation status of the penultimate threonine at the C-terminus of the proton pumps,\u0026nbsp;membranes were blocked with 5% [w/v] skim milk in far-western buffer (20 mM MES, 130 mM NaCl, 10 mM MgSO\u003csub\u003e4\u003c/sub\u003e and 100 \u0026micro;M CaCl\u003csub\u003e2,\u003c/sub\u003e pH 6.5) for 1h at room temperature. Then membranes were transferred into 10 ml far-western buffer containing 40 \u0026mu;g RGS-His epitope tagged 14-3-3 (GF14\u0026oslash;) protein\u003csup\u003e23\u003c/sup\u003e and 5 \u0026micro;M fusicoccin (dissolved in 96% ethanol, Santa Cruz Biotechnology) for 1h at room temperature. 14-3-3 protein binding was detected using a monoclonal anti-RGS-6x-His antibody (1:1000; Qiagen)\u0026nbsp;followed by anti-mouse-HRP\u0026nbsp;(1:10000;\u0026nbsp;Millipore) as the primary and secondary antibodies, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInsect feeding assays\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eManduca sexta\u003c/em\u003e eggs were collected and kept in a climate chamber\u0026nbsp;at 16 h/26\u0026deg;C and 8 h/24\u0026deg;C\u0026nbsp;light-dark cycle until the larvae hatched. Two freshly-hatched\u0026nbsp;neonates were placed on the 2\u003csup\u003end\u003c/sup\u003e true leaf (from bottom to top) of 4-week-old tomato plants and enclosed in white organza bags. The developing larvae were moved onto younger leaves when the tissue was consumed. Larval mass was measured every two days until all leaves were consumed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of proteinase inbibitor (PI-II) activity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteinase inhibitor activity was assayed in tomato leaf extracts as soybean trypsin inhibitor (STI) equivalents using a radial diffusion assay as previously described\u003csup\u003e53\u003c/sup\u003e. Briefly,\u0026nbsp;leaf disc samples\u0026nbsp;(150 mg) were collected into 2 mL screw-cap tubes containing 0.6 g ZrO\u003csub\u003e2\u003c/sub\u003e beads (2.8-3.2 mm; M\u0026uuml;hlmeier, Germany) and flash-frozen in liquid nitrogen. Samples were homogenised in 500 \u0026micro;l extraction buffer (50 g/l PVPP, 18.6 g/l Na\u003csub\u003e2\u003c/sub\u003eEDTA, 5 g/l Na-diethyldithiocarbamate-trihydrate, 2 g/l phenylthiourea in 0.1 M Tris-HCl, pH 7.6 for 150 \u0026micro;g of tissue) in a Fisherbrand\u0026trade; Bead Mill 24 homogenizer at 4 m/s for 2 x 30 s. The extract was cleared by centrifugation (15 min, 16100 g, 4\u0026deg;C). The supernatant was filled into \u0026oslash; 0.4 mm-wells punched into 12 x 12 cm plates containing 25 ml of 1.8% [w/v] agar with 2 mg bovine trypsin (Sigma-Aldrich) in 0.1 M Tris-HCl, pH 7.6). After 18 hours at 4\u0026deg;C, 25 ml substrate solution (6 mg \u003cem\u003eN\u003c/em\u003e-acetyl-\u003cem\u003eDL\u003c/em\u003e-phenylalanine-naphthyl ester, 12 mg Fast Blue B salt in 0.1 M Tris/20% [v/v] dimethyl formamide) were added to stain for trypsin activity during 1 h at 37 \u0026deg;C. The diameter of the clear inhibition zones surrounding each well was used to calculate\u0026nbsp;trypsin protease inhibitor\u0026nbsp;activity using a standard curve with soybean trypsin inhibitor (Sigma-Aldrich) as the reference.\u0026nbsp;16\u0026nbsp;plants\u0026nbsp;were\u0026nbsp;analyzed per genotype in\u0026nbsp;two\u0026nbsp;technical replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRhizosphere acidification\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand alkalinization\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eassays.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive to six-day-old wild-type and \u003cem\u003epll2\u003c/em\u003e tomato seedlings grown on ATS plates were transferred onto pH indicator plates containing 0.002 % [w/v] bromocresol purple (BCP) in water-ager (0.5 % [w/v] agar, pH 6.5 adjusted with KOH). Pictures were taken after 24 h of incubation. To monitor systemin-induced alkalinization of the rhizosphere, seedlings were transferred from ATS plates to indicator plates adjusted to pH 5.5 with acetic acid. They were then sprayed with 1 \u0026mu;M systemin or water as the control. Pictures were taken before and 30 minutes after application.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanalysis\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetic trees\u0026nbsp;of\u0026nbsp;\u003cem\u003eSolanum lycopersicum\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e proteins in the\u0026nbsp;P-type H\u003csup\u003e+\u003c/sup\u003e-ATPase and\u0026nbsp;POLTERGEIST-LIKE\u0026nbsp;families were generated\u0026nbsp;using\u0026nbsp;PhyloGenes\u0026nbsp;v4.1(\u003ca href=\"http://www.phylogenes.org\"\u003ewww.phylogenes.org\u003c/a\u003e)\u003csup\u003e54\u003c/sup\u003e.\u0026nbsp;SlLHA1 (Solyc03g113400), SlLHA4\u0026nbsp;(Solyc07g017780), and SlPLL2 (Solyc06g076100)\u0026nbsp;are\u0026nbsp;highlighted in red.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal microscopy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubcellular localization of GFP-fusion proteins transiently expressed\u0026nbsp;in\u0026nbsp;\u003cem\u003eN. benthamiana\u003c/em\u003e was\u0026nbsp;imaged on a Zeiss LSM900 confocal microscope. The excitation\u0026nbsp;and emission\u0026nbsp;wavelengths were\u0026nbsp;488 and 509\u0026nbsp;nm for GFP, and\u0026nbsp;506\u0026nbsp;and\u0026nbsp;751\u0026nbsp;nm for FM4-64, respectively. Bimolecular fluorescence complementation (BiFC) in\u0026nbsp;\u003cem\u003eN. benthamiana\u003c/em\u003e was\u0026nbsp;analyzed\u0026nbsp;on a Zeiss LSM700 confocal microscope.\u0026nbsp;Fluorescence signals were recorded for GFP (excitation\u0026nbsp;488\u0026nbsp;nm, emission 518 nm) and YFP (excitation\u0026nbsp;488\u0026nbsp;nm, emission 518 nm). Multiple leaves of at least two independent plants were analyzed.\u0026nbsp;Figures were prepared using\u0026nbsp;open source\u0026nbsp;software Fiji\u003csup\u003e55\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical tests in phosphoproteomics\u0026nbsp;were performed using R packages (Limma, factoextra, hypeR). Details of other statistical analyses are indicated in the figure legends.\u0026nbsp;Unpaired two tailed Student\u0026rsquo;s\u003cem\u003e\u0026nbsp;t\u003c/em\u003e-test and one-way ANOVA with Tukey\u0026rsquo;s multiple comparisons test were performed in GraphPad Prism 9.0 (GraphPad Software, Inc.).  For all box plots, boxes range from 25th to 75th percentiles with the splitting line at the median. Whiskers extend to the minimum and maximum values if lower than 1.5x interquartile range.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank Claudia Oecking (University of T\u0026uuml;bingen) and Xu Wang (University of Hohenheim) for helpful discussions, and Georg Felix (University of T\u0026uuml;bingen) and Xu Wang for the 35S::SYR1:GFP and 35S::SYR1 expression constructs, respectively. Technical assistance by Bianca Bukowski and Latisha Alleyne-Lafleur is gratefully acknowledged. Funding: The work was supported by the Deutsche Forschungsgemeinschaft [SFB 1101 to ASc and ASti (project D06)].\u003c/p\u003e\u003ch2\u003eCode availability\u003c/h2\u003e \u003cp\u003eR Scripts for phosphoproteomic analysis and the linear regression plot of Fig.\u0026nbsp;2a are available at GitHub (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/shibalili/systemin-project\u003c/span\u003e\u003cspan address=\"https://github.com/shibalili/systemin-project\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All bioinformatic tools used in this study are cited in the Methods section.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e partner repository with the dataset identifier PXD054229 Source data are provided with this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: ASc, ASti. Investigation: RL, FHA. Visualization: RL, ASc. Funding acquisition: ASc, ASti. Supervision ASti, ASte. Writiting \u0026ndash; original draft: RL, ASc. Writing \u0026ndash; review and editing: all authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtended data is available for this paper at\u0026nbsp;https://doi.org...\u003c/p\u003e\n\u003cp\u003eSupplementary information: The online version contains supplementary material available at https://doi.org....\u003c/p\u003e\n\u003cp\u003eCorrespondence and requests for materials should be addressed to [email protected] or [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePearce, G., Strydom, D., Johnson, S. \u0026amp; Ryan, C. A. A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins. Science 253, 895\u0026ndash;898 (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchilmiller, A. L. \u0026amp; Howe, G. A. Systemic signaling in the wound response. Curr. Opin. Plant Biol. 8, 369\u0026ndash;377 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, L. \u003cem\u003eet al.\u003c/em\u003e The systemin receptor SYR1 enhances resistance of tomato against herbivorous insects. Nature Plants 4, 152\u0026ndash;156 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeloshistov, R. E. \u003cem\u003eet al.\u003c/em\u003e Phytaspase-mediated precursor processing and maturation of the wound hormone systemin. New Phytol. 218, 1167\u0026ndash;1178 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGust, A. A., Pruitt, R. \u0026amp; N\u0026uuml;rnberger, T. Sensing danger: key to activating plant immunity. Trends Plant Sci. 2, 779\u0026ndash;791 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanfermeijer, F. C., Staal, M., Malinowski, R., Stratmann, J. W. \u0026amp; Elzenga, J. T. M. Micro-electrode flux estimation confirms that the \u003cem\u003eSolanum pimpinellifolium cu3\u003c/em\u003e mutant still responds to systemin. Plant Physiol. 146, 129\u0026ndash;139 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFelix, G. \u0026amp; Boller, T. Systemin induces rapid ion fluxes and ethylene biosynthesis in \u003cem\u003eLycopersicon peruvianum\u003c/em\u003e cells. Plant J. 7, 381\u0026ndash;389 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoyen, C., Hammond-Kosack, K. E., Jones, J., Knight, M. R. \u0026amp; Johannes, E. Systemin triggers an increase of cytoplasmic calcium in tomato mesophyll cells: Ca\u003csup\u003e2+\u003c/sup\u003e mobilization from intra- and extracellular compartments. Plant Cell Environ 21, 1101\u0026ndash;1111 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoller, T. Chemoperception of microbial signals in plant cells. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 189\u0026ndash;214 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEscocard de Azevedo Manh\u0026atilde;es, A. M., Ortiz-Morea, F. A., He, P. \u0026amp; Shan, L. Plant plasma membrane-resident receptors: Surveillance for infections and coordination for growth and development. J. Int. Plant Biol. 63, 79\u0026ndash;101 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZipfel, C. \u003cem\u003eet al.\u003c/em\u003e Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125, 749\u0026ndash;760 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKunze, G. \u003cem\u003eet al.\u003c/em\u003e The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16, 3496\u0026ndash;3507 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWasternack, C. Jasmonates: An update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann. Bot. 100, 681\u0026ndash;697 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaj Ahmad, F., Wu, X., Stintzi, A., Schaller, A. \u0026amp; Schulze, W. X. The systemin signaling cascade as derived from time course analyses of the systemin-responsive phosphoproteome. Mol. Cell. Proteomics 18, 1526\u0026ndash;1542 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSobol, G., Chakraborty, J., Martin, G. B. \u0026amp; Sessa, G. The emerging role of PP2C phosphatases in tomato immunity. Mol Plant Microbe In 35, 737\u0026ndash;747 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchaller, A. \u0026amp; Oecking, C. Modulation of plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell 11, 263\u0026ndash;272 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumari, A., Ch\u0026eacute;telat, A., Nguyen, C. T. \u0026amp; Farmer, E. E. \u003cem\u003eArabidopsis\u003c/em\u003e H\u003csup\u003e+\u003c/sup\u003e-ATPase AHA1 controls slow wave potential duration and wound-response jasmonate pathway activation. \u003cem\u003eProc. Natl. Acad. Sci USA\u003c/em\u003e 116, 20226\u0026ndash;20231 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJulien, J. L. \u0026amp; Frachisse, J. M. Involvement of the proton pump and proton conductance change in the wave of depolarization induced by wounding in \u003cem\u003eBidens pilosa\u003c/em\u003e. Can. J. Bot. 70, 1451\u0026ndash;1458 (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchaller, A. \u0026amp; Frasson, D. Induction of wound response gene expression in tomato leaves by ionophores. Planta 212, 431\u0026ndash;435 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaruta, M., Gray, W. M. \u0026amp; Sussman, M. R. Regulation of the plasma membrane proton pump (H\u003csup\u003e+\u003c/sup\u003e-ATPase) by phosphorylation. Curr Opin Plant Biol 28, 68\u0026ndash;75 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalhof, J., Pedersen, J. T., Fuglsang, A. T. \u0026amp; Palmgren, M. Plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase regulation in the center of plant physiology. Mol. Plant 9, 323\u0026ndash;337 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSvennelid, F. \u003cem\u003eet al.\u003c/em\u003e Phosphorylation of Thr-948 at the C terminus of the plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase creates a binding site for the regulatory 14-3-3 protein. Plant Cell 11, 2379\u0026ndash;2391 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJahn, T. \u003cem\u003eet al.\u003c/em\u003e The 14-3-3 protein interacts directly with the C-terminal region of the plant plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase. Plant Cell 9, 1805\u0026ndash;1814 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEisenhaber, F. \u003cem\u003eet al.\u003c/em\u003e Prediction of lipid posttranslational modifications and localization signals from protein sequences: big-Pi, NMT and PTS1. Nucleic Acids Res 31, 3631\u0026ndash;3634 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuglsang, A. T. \u003cem\u003eet al.\u003c/em\u003e Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+-ATPase by preventing interaction with 14-3-3 protein. Plant Cell 19, 1617\u0026ndash;1634 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbez, E., D\u0026uuml;nser, K., Gaidora, A., Lendl, T. \u0026amp; Busch, W. Auxin steers root cell expansion via apoplastic pH regulation in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e 114, E4884-E4893 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeFalco, T. A. \u003cem\u003eet al.\u003c/em\u003e A conserved module regulates receptor kinase signalling in immunity and development. Nature Plants (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark, C.-J. \u003cem\u003eet al.\u003c/em\u003e Rice XB15, a protein phosphatase 2C, negatively regulates cell death and XA21-mediated innate immunity. PLOS Biology 6, e231 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpartz, A. K. \u003cem\u003eet al.\u003c/em\u003e SAUR Inhibition of PP2C-D phosphatases activates plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPases to promote cell expansion in Arabidopsis. Plant Cell 26, 2129\u0026ndash;2142 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRen, H., Park, M. Y., Spartz, A. K., Wong, J. H. \u0026amp; Gray, W. M. A subset of plasma membrane-localized PP2C.D phosphatases negatively regulate SAUR-mediated cell expansion in Arabidopsis. PLoS genetics 14, e1007455 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin, W. \u003cem\u003eet al.\u003c/em\u003e TMK-based cell-surface auxin signalling activates cell-wall acidification. Nature 599, 278\u0026ndash;282 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNuhse, T. S., Bottrill, A. R., Jones, A. M. E. \u0026amp; Peck, S. C. Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant J. 51, 931\u0026ndash;940 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaruta, M., Sabat, G., Stecker, K., Minkoff, B. B. \u0026amp; Sussman, M. R. A peptide hormone and its receptor protein kinase regulate plant cell expansion. Science 343, 408\u0026ndash;411 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, X., Li, R., Stintzi, A. \u0026amp; Schaller, A. Automated real-time monitoring of extracellular pH to assess early plant defense signaling. in \u003cem\u003ePlant Peptide Hormones and Growth Factors\u003c/em\u003e Vol. 2731 \u003cem\u003eMeth. Mol. Biol.\u003c/em\u003e (ed A. Schaller) 169\u0026ndash;178 (Humana, 2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCox, J. \u003cem\u003eet al.\u003c/em\u003e Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 13, 2513\u0026ndash;2526 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eR.CoreTeam. R: A language and environment for statistical computing.. \u003cem\u003eR Foundation for Statistical Computing\u003c/em\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWickham, H. \u0026amp; al., e. Welcome to the Tidyverse. J Open Source Softw 4, 1668 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWickham, H. \u003cem\u003eggplot2: Elegant graphics for data analysis\u003c/em\u003e. (Springer-Verlag New York, 2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRitchie, M. E. \u003cem\u003eet al.\u003c/em\u003e limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43, 20 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStekhoven, D. J. \u0026amp; B\u0026uuml;hlmann, P. MissForest\u0026mdash;non-parametric missing value imputation for mixed-type data. Bioinformatics 28, 112\u0026ndash;118 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTibshirani, R., Walther, G. \u0026amp; Hastie, T. Estimating the number of clusters in a data set via the gap statistic. Journal of the Royal Statistical Society Series B: Statistical Methodology 63, 411\u0026ndash;423 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKassambara, A. \u0026amp; Mundt, F. factoextra: Extract and visualize the results of multivariate data analyses (R package version 1.0.7). (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwacke, R. \u003cem\u003eet al.\u003c/em\u003e MapMan4: A refined protein classification and annotation framework applicable to multi-omics data analysis. Mol. Plant 12, 879\u0026ndash;892 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTheTomatoGenomeConsortium. The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485, 635\u0026ndash;641 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFederico, A. \u0026amp; Monti, S. hypeR: an R package for geneset enrichment workflows. Bioinformatics 36, 1307\u0026ndash;1308 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGleave, A. P. A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203\u0026ndash;1207 (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, X., Robles Luna, G., Arighi, C. N. \u0026amp; Lee, J.-Y. An evolutionarily conserved motif is required for Plasmodesmata-located protein 5 to regulate cell-to-cell movement. Commun Biol 3, 291 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXing, H.-L. \u003cem\u003eet al.\u003c/em\u003e A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology 14, 327 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBosch, M. \u003cem\u003eet al.\u003c/em\u003e Jasmonic acid and its precursor 12-oxophytodienoic acid control different aspects of constitutive and induced herbivore defenses in tomato. Plant Physiol. 166, 396\u0026ndash;410 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCedzich, A. \u003cem\u003eet al.\u003c/em\u003e The protease-associated (PA) domain and C-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase 3 (SlSBT3). J. Biol. Chem. 284, 14068\u0026ndash;14078 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReichardt, S., Stintzi, A. \u0026amp; Schaller, A. Assay for Phytaspase-mediated Peptide Precursor Cleavage Using Synthetic Oligopeptide Substrates. \u003cem\u003ebio-protocol\u003c/em\u003e 13, e4608 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, R., Schaller, A. \u0026amp; Stintzi, A. Quantitative measurement of pattern-triggered ROS burst as an early immune response in tomato. in \u003cem\u003ePlant Peptide Hormones and Growth Factors\u003c/em\u003e Vol. 2731 \u003cem\u003eMeth. Mol. Biol.\u003c/em\u003e (ed A. Schaller) 157\u0026ndash;167 (Humana, 2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBandoly, M., Grichnik, R., Hilker, M. \u0026amp; Steppuhn, A. Priming of anti-herbivore defence in Nicotiana attenuata by insect oviposition: herbivore-specific effects. Plant, Cell \u0026amp; Environment 39, 848\u0026ndash;859 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, P. \u003cem\u003eet al.\u003c/em\u003e PhyloGenes: An online phylogenetics and functional genomics resource for plant gene function inference. Plant Direct 4, e00293 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchindelin, J. \u003cem\u003eet al.\u003c/em\u003e Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676\u0026ndash;682 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez-Riverol, Y. \u003cem\u003eet al.\u003c/em\u003e The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucl. Acids Res. 50, D543-D552 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4919676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4919676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Systemin, the first signaling peptide identified in plants, mediates induced resistance against insect herbivores in tomato\u003csup\u003e1\u003c/sup\u003e. Initially, systemin was perceived as a hormone-like, long-distance messenger that triggers systemic defense responses far from the site of insect attack. It was later found to rather act as a phytocytokine, amplifying the local wound response for the production of downstream signals that activate defense gene expression in distant tissues\u003csup\u003e2\u003c/sup\u003e. Systemin perception and signaling rely on the systemin receptor SYR1\u003csup\u003e3\u003c/sup\u003e. However, the specifics of SYR1-dependent signaling and how systemin signaling differs from other phytocytokine signaling pathways remain largely unknown. Here, we report that systemin activates the poltergeist-like phosphatase PLL2 in a SYR1-dependent manner. PLL2, in turn, regulates early systemin responses at the plasma membrane, including the rapid inhibition of proton pumps through the dephosphorylation of their regulatory C-termini. PLL2 was found to be essential for downstream defense gene induction, ultimately contributing to insect resistance.","manuscriptTitle":"Poltergeist-Like 2 (PLL2)-dependent activation of the wound response distinguishes systemin from other immune signaling pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-24 05:00:30","doi":"10.21203/rs.3.rs-4919676/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-plants","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nplants","sideBox":"Learn more about [Nature Plants](http://www.nature.com/nplants/)","snPcode":"","submissionUrl":"","title":"Nature Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8cc94b24-d041-484e-8c8a-eb0363ff6a64","owner":[],"postedDate":"October 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38297417,"name":"Biological sciences/Plant sciences/Plant immunity/Pattern recognition receptors in plants"},{"id":38297418,"name":"Biological sciences/Plant sciences/Plant stress responses/Herbivory"},{"id":38297419,"name":"Biological sciences/Plant sciences/Plant signalling"},{"id":38297420,"name":"Biological sciences/Plant sciences/Plant stress responses/Wounding"},{"id":38297421,"name":"Biological sciences/Plant sciences/Plant stress responses/Biotic"}],"tags":[],"updatedAt":"2025-07-05T07:10:14+00:00","versionOfRecord":{"articleIdentity":"rs-4919676","link":"https://doi.org/10.1038/s41477-025-02040-7","journal":{"identity":"nature-plants","isVorOnly":false,"title":"Nature Plants"},"publishedOn":"2025-07-04 04:00:00","publishedOnDateReadable":"July 4th, 2025"},"versionCreatedAt":"2024-10-24 05:00:30","video":"","vorDoi":"10.1038/s41477-025-02040-7","vorDoiUrl":"https://doi.org/10.1038/s41477-025-02040-7","workflowStages":[]},"version":"v1","identity":"rs-4919676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4919676","identity":"rs-4919676","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}