Poltergeist-Like 2 (PLL2)-dependent activation of herbivore defence distinguishes systemin from other immune signalling pathways

Abstract

Systemin, the first signalling peptide identified in plants, mediates induced resistance against insect herbivores and necrotrophic pathogens in tomato^1-3. Initially, systemin was conceived as a hormone-like, long-distance messenger that triggers systemic defence 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 defence gene expression in distant tissues⁴. Systemin perception and signalling rely on the systemin receptor SYR1⁵. However, the specifics of SYR1-dependent signalling and how systemin signalling differs from other immune signalling 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 dephosphorylation of their regulatory C-termini. PLL2 was found to be essential for downstream defence gene induction, ultimately contributing to insect resistance.

Fig. 1. The systemin response is characterized by transient dephosphorylation of cellular proteins at 1 min after treatment.

a, Extracellular alkalinization is an early hallmark of systemin and pattern-triggered immune signalling. The pH of the culture medium was recorded after addition of 10 nM systemin or 20 nM flg22 to wild-type and syr1 cells. Data points represent the mean ± standard error (s.e.) of six independent experiments. WT, wild type. b, The systemin-induced phospho-proteomic response of wild-type cells is lost in syr1. PCA is shown for phospho-site datasets (average of six biological replicates for each genotype and time point after systemin treatment). c,d, K-means clustering of phospho-sites (P-sites) according to their temporal changes in intensity identified five time profiles (clusters) between the wild-type and syr1 cell cultures (d), with the number of sites per genotype and cluster summarized in c. A transient drop in intensity is frequently observed in wild type but not in syr1 (clusters 2 and 3 in c and d). e, Phosphosites that belong to clusters 2 and 3 in wild type distribute to other clusters in syr1. The black lines in d and e show the median of phospho-site intensity for each cluster. f, Systemin-induced changes in phosphorylation at Ser151, Ser142 and Ser160 of SlPLL2 in wild-type cells. The experiment included six biological replicates. 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 1.5× interquartile range. For Ser151, which has data for each time point, medians are connected by a trend line. Adjusted P values are shown for time points significantly different from t = 0 min (two-tailed ANOVA with Dunnett’s multiple comparison test). Source data

Fig. 2. The dephosphorylated, active form of SlPLL2 inhibits tomato P-type H⁺-ATPases.

a,b, SlPLL2 activity depends on its phosphorylation status. Equal amounts of SlPLL2^WT, phospho-dead SlPLL2^3A, phospho-mimetic SlPLL2^3D and free GFP (negative control) purified from agro-infiltrated N. benthamiana leaves (Extended Data Fig. 3) were used in activity assays based on the colorimetric quantification of inorganic phosphate (Pi) release from a synthetic phospho-peptide substrate (pLHA1, 100 µM). Data represent the mean ± s.d. of six independent experiments. Different letters indicate significant differences at P < 0.05 (two-tailed ANOVA followed by Tukey test). OD₆₀₀, optical density at 600 nm. a, Progress curves of phosphate release. b, Activity of SlPLL2 variants in pmol phosphate per hour. c,d, Co-IP assay of SlPLL2 interaction with LHA1 and LHA4. SlPLL2 phospho-variants were co-expressed as GFP fusions with the Flag-tagged regulatory (R) domain of LHA4 (c) or LHA1 (d) in N. benthamiana leaves. Total protein extracts from leaves treated with 100 nM systemin or flg22 were immunoprecipitated with anti-Flag agarose beads, then detected with anti-GFP on western blots. Co-IP assays were performed in triplicate for c and in duplicate for d. e, Growth assays showing SlPLL2-mediated regulation of H⁺-ATPase activity in yeast. Yeast strain RS-72 was (co-)transformed with expressions constructs for tomato H⁺-ATPases LHA1 (two lower panels) or LHA4 (two upper panels) and SlPLL2 phospho-variants in different combinations. Cells were spotted in two dilutions (OD₆₀₀ = 0.1 or 0.01) on control medium (Gal) and on selective medium (Glu), where growth depends on LHA1 or LHA4 activity. Results are representative of three biological replicates. f, Western blot overlay monitoring the phosphorylation status of the regulatory threonine of LHA1 and LHA4. Microsomal fractions (100 µg) of yeast cultures (co-)expressing LHA1 (lower panel) or LHA4 (upper panel) with SlPLL2 variants were separated by SDS–PAGE and analysed on western blots using an antiserum against the Arabidopsis H⁺-ATPase (AHA2); 14–3–3 protein binding distinguishes the penultimate threonine in its phosphorylated state from the non-phosphorylated state. Coomassie brilliant blue-stained gels (CBB) are shown as loading control. Experiments were performed three times with similar results. In e and f, equal expression of PLL2 variants in yeast was confirmed by western blot analysis (Supplementary Fig. 1). Source data

Fig. 3. Systemin responses depend on SlPLL2.

a, Loss of PLL2 function leads to increased proton extrusion in pll2 compared to wild-type seedlings. Extracellular pH was monitored in the rhizosphere of seedlings transferred from ATS medium to bromocresol purple (BCP) pH indicator plates at pH 6.5. Changes in medium colour were recorded after 24 and 48 h. b, Systemin-induced increase in extracellular pH depends on PLL2. After transfer to BCP indicator plates at pH 5.5, seedlings were sprayed with systemin (1 μM) or water (mock). Medium colour was recorded at 0 and 30 min after spraying. Sys, systemin. In a and b, similar results were obtained in three independent experiments. c, Root growth is enhanced in pll2 compared to wild-type seedlings grown on ATS medium (n = 31 for wild type and 28 for pll2). d, Systemin-induced growth inhibition is impaired in pll2. Root and hypocotyl growth inhibition in pll2 and wild-type seedlings is shown as the length of systemin-treated seedlings divided by the average length on control plates (n = 29 seedlings of each genotype). In c and d, two-tailed Student’s t-test was used to test for statistically significant differences; P values are indicated. The experiments were repeated three times with similar results. e, Systemin-induced increase in extracellular pH depends on proton pump inactivation. Wild-type seedlings grown on ATS medium were sprayed with 10 µM fusicoccin (+FC) or water (−FC). After 15 min, seedlings were transferred to BCP indicator plates at pH 5.5 and sprayed with systemin (1 µM). Medium colour was recorded at 0 and 30 min after systemin treatment. Similar results were obtained in three independent experiments. f, Systemin-triggered MAPK activation is reduced in pll2. Extracts (30 µg of protein) of systemin (1 μM)-treated plants were analysed by immunoblotting using an anti-pERK antibody. A Coomassie brilliant blue-stained gel is shown as loading control (CBB). Representative results are shown for one of two independent experiments. g,h, ROS production is impaired in pll2. ROS production was analysed in leaf discs of wild-type and pll2 plants treated with 30 nM systemin (g) or flg22 (h). Progress curves show the mean ± s.e. of 31 (pll2) and 32 (wild-type) plants. Results are representative for three independent experiments with three different pll2 alleles (Extended Data Fig. 6). i,j, The systemin-induced ROS burst correlates with PLL2 activity. The ROS burst was induced by adding 30 nM systemin (i) or flg22 (j) to leaf discs of N. benthamiana plants expressing Spot-tagged SlPLL2^WT, SlPLL2^3A and SlPLL2^3D variants or only the P19 vector as control. SYR1-eGFP was co-expressed in i to enable systemin perception in N. benthamiana. Equal expression of SYR1-eGFP in the presence of different SlPLL2 variants was confirmed by western blot analysis (Supplementary Fig. 2). Data represent the mean ± s.e. of n = 39 (in i) or 40 (in j) plants. The experiment was repeated using GFP-tagged SlPLL2 variants with similar results. k,l, Induction of PI-II expression by systemin and wounding is impaired in pll2 compared to wild-type plants. RT-qPCR was performed with RNA extracted from leaf tissue collected before and 1, 3 and 6 h after systemin (1 µM) treatment (k) or wounding (l). Relative PI-II expression is shown as fold change over the untreated control (n = 12 biological replicates; two-tailed Student’s t-test; P values are indicated). m,n, Herbivore defence is impaired in pll2 compared to wild-type plants. Freshly hatched M. sexta were placed on the second oldest leaf of 4-week-old plants and, when all leaf material had been consumed, transferred to progressively younger leaves. m, After 5 days, proteinase inhibitor (PI) activity was analysed in systemic (unwounded) leaves. PI activity is shown as soybean trypsin inhibitor (STI) equivalents per gram fresh weight (FW) for n = 16 plants. n, Mass of n = 37 M. sexta larvae after 2 weeks of feeding. Scale bar, 5 cm. The experiment was repeated twice with two independent pll2 lines, showing similar results. In m and n, two-tailed Student’s t-test was used for pairwise comparisons; P values are indicated. Box plots in c, d and k–n range from the 25th to 75th percentiles with the splitting line at the median. Whiskers extend to the minimum and maximum values if lower than 1.5 × interquartile range. Source data

Extended Data Fig. 1. Design of the phospho-proteomics experiment.

a, Phospho-proteomics workflow. Samples were harvested from WT and syr1 cell cultures before and at 5 time points after systemin treatment in six biological replicates, resulting in a total of 72 samples. From each sample, the microsomal membrane fraction was extracted and digested with trypsin and LysC. After phospho-peptide enrichment, samples were analyzed by LC-MS/MS. Raw files were transferred to MaxQuant for peptide quantification using the tomato proteome database ITAG3.2 as the reference. b, Hierarchical clustering of the six biological replicates clearly separated the two genotypes.

Extended Data Fig. 2. Selection of PLL2 as a candidate phosphatase specifically involved in systemin signalling.

a, The K-means clustering algorithm requires a predefined number of clusters K that is chosen to best describe the structure of the data. Gap statistics showed that the cluster structure showed maximum distance from the random uniform distribution of points with the optimal number of K = 5 clusters. 10 was set as the upper limit for K and 100 bootstrapping runs were performed in fviz_nbclust (method = “gap_stat”). Data points show the mean of 100 bootstrapping runs ± SE. b, Protein phosphatases represented in this phosphoproteome dataset belonged to four different families: metallo-dependent protein phosphatases (PPM)/ type 2C protein phosphatases (PP2C), phosphoprotein phosphatases (PPP), protein tyrosine phosphatases (PTP) and aspartate (Asp)-dependent phosphatases. Intensity of shading is used to indicate the number of detected phospho-sites with darker colors indicating more phospho-sites for a given protein. Most phospho-sites were found in POLTERGEIST-LIKE phosphatases (POL, boxed in orange). c, Phylogenetic tree of POL family proteins from Arabidopsis and tomato. The tree was generated by PhyloGenes (v4.1) based on GIGA algorithm. SlPLL2 (highlighted in red) is located in a sister clade to Arabidopsis PLL2 – PLL5. d, SlPLL2 has three phospho-sites that show a transient drop in intensity at 1 min after systemin treatment (cluster 2). Cluster distribution is shown for the phospho-sites of PPM/PP2C family phosphatases. The number of phospho-sites in each cluster is reflected by the intensity of color shading and size of the dots. SlPLL2 (Solyc06g076100) stands out as all three differentially phosphorylated sites belong to cluster 2.

Extended Data Fig. 3. Quantification of SlPLL2 variants in protein extracts used for phosphatase activity assays.

As shown in Fig. 2a,b. SlPLL2^WT, SlPLL2^3A and SlPLL2^3D were expressed as C-terminal GFP fusion proteins in N. benthamiana, and purified from leaf extracts by GFP-trap (Chromotek). Abundance of the GFP fusion proteins was analyzed by anti-GFP immunoblotting. In each of the six independent experiments, similar expression levels were observed for the three SlPLL2 variants. Source data

Extended Data Fig. 4. Identification of plasma membrane proton pumps LHA1 and LHA4 as candidate substrates of SlPLL2.

a, over-representation analysis of biological processes in the five clusters. The proteins represented by the phospho-sites in each of the five clusters were subjected to MapMan over-representation analysis. MapMan bins of biological processes that were significantly (P < 0.05) enriched are plotted according to cluster number. The size of the dots reflects the number of phospho-sites for each bin, color shading indicates P value (hypergeometric test, one-sided). Bins related to solute transport (bin 24) are enriched in clusters 2 and 3 including LHA1 and LHA4. b, phylogenetic analysis of tomato and Arabidopsis P-type H⁺-ATPases. The tree was generated by PhyloGenes (v4.1) based on GIGA algorithm. LHA1 and LHA4 are highlighted in red. c, d normalized intensity of phosphorylation of the regulatory threonine T951 of LHA4 (c, Solyc07g017780) and T955 of LHA1 (d, Solyc03g113400) is shown in a time series after systemin treatment of the S. peruvianum wild-type (WT; red) and syr1 (blue) cell cultures in six biological replicates. The trendline connects the mean intensity at each time point.

Extended Data Fig. 5. LHA1/4 and SlPLL2 interact at the plasma membrane.

a, SlPLL2 localizes to the plasma membrane irrespective of the phosphorylation status of S142-S151-S160. SlPLL2-GFP (WT, 3A and 3D mutants) were transiently expressed in N. benthamiana leaves. Leaf pieces were incubated in 10 µM FM4-64 (plasma membrane marker) 15 min before confocal imaging using a Zeiss LSM980 microscope in the red (FM4-64) and green (GFP) channels, respectively; scale bar = 10 µm. b, Interaction of LHA1/4 and SlPLL2 is indicated by bimolecular fluorescence complementation (BiFC). Tomato H⁺-ATPases (LHA1 and LHA4) fused with nYFP, and SlPLL2 with cYFP were transiently expressed in N. benthamiana leaves. LHA1 and LHA4 fused with full-length YFP, and SlPLL2 with GFP were used as positive controls. Negative controls included leaves expressing LHA1-nYFP or LHA4-nYFP together with SERK3B-cYFP, and SlPLL2-cYFP co-expressed with SYR1-nYFP; scale bar = 50 μm. a, and b, the expriments were repeated at least three times with similar results. c, All fusion proteins used in the BiFC experiment are expressed in N. benthamiana leaves. Anti-GFP western blot analysis of protein extracts from N. benthamina leaves used in the BiFC experiment shown in (b). Colored asterisks mark the bands of the corresponding fusion proteins. This control blot was done for one representative experiment.

Extended Data Fig. 6. Three independent pll2 alleles were used in this study.

Sequence analysis revealed three independent CRISPR/Cas9-generated mutations at the 1st guide RNA resulting in the addition of one, and deletion of 1 or 595 base pairs (boxed in red) from the SlPLL2 locus. These mutations resulted in truncated proteins of 145, 147, and 219 amino acids, respectively. The PAM sequence is indicated in green; the Cas9 cleavage site is located 3 bp upstream of the PAM sequence (red arrow).

Extended Data Fig. 7. Regulation of PLL2 by dephosphorylation is only observed after systemin treatment and is not involved in chitin signalling.

a, Chitin-induced ROS production is impaired in pll2. ROS production was analyzed in leaf discs of 4-week-old tomato WT (n = 31) and pll2 (n = 30) plants treated with 50 µg/ml chitin. Progress curves show the mean ± SE. b, Tomato PLL2 interferes with chitin-induced ROS production in N. benthamiana irrespective of its activity. The ROS burst was analyzed in leaf discs of N. benthamiana plants expressing SlPLL2^WT, SlPLL2^3A and SlPLL2^3D variants, or the P19 vector only as a control. Data represent the mean ± SE of n = 40 plants for each expression construct. c, Rapid and transient dephosphorylation of Ser151 is systemin specific and not observed in response to flg22 or chitin. Normalized intensity of the phospho-peptide comprising the Ser151 phospho-site is shown in a time series after elicitor treatment of the S. peruvianum cell culture in six biological replicates. The trendline connects the mean intensity at each time point. Source data

Extended Data Fig. 8. REF1 responses do not depend on PLL2.

a, there is no ROS burst in response to REF1 treatment. ROS production was analyzed in leaf discs of 4-week-old tomato WT and pll2 plants treated with 30 nM systemin (n = 12) or REF1 (n = 8). Progress curves show the mean ± SE. b, REF1-triggered MAPK activation is not impaired in pll2. Leaf tissue was harvested from three-week-old plants in a time series after REF1 (1 μM) treatment. Extracts (30 µg of protein) were analyzed by immunoblotting using an anti-pERK antibody. A Coomassie Brilliant Blue-stained gel is shown as loading control (CBB). Representative results are shown for one of two independent experiments c, PI-II expression is induced by REF1 to somewhat higher levels in pll2 compared to WT. Leaf tissue of three-week-old plants was harvested at 1, 2, and 6 h after REF1 (1 µM) treatment for RNA extraction and RT-qPCR analysis. Relative PI-II expression is shown as fold change over the untreated control. Results are shown as box plots for n = 3 biological replicates; whiskers extend from the median to maximum and minimum values. Two-tailed Student’s t-test was used to compare induction levels between WT and pll2; P values are indicated). Source data

Extended Data Fig. 9. Domain structure of PLL2 and conserved phosphorylation sites in its regulatory N-terminus.

Regulatory phosphorylation sites are mostly conserved between tomato PLL2 and Arabidopsis PLL4/5 but differ in function. The sequence alignment shows seven predicted phosphorylation sites in the SXXL/I sequence context (green) in the N-terminal regulatory domain of PLLs. Six of these sites (mutated en block) were shown to be important for the AtPLL4/5 mediated inhibition of pattern-triggered immunity. At least one of these sites, Ser151, and possibly serines 142 and 160 of SlPLL2, were found here to regulate phosphatase activity for the activation of herbivore defence signalling.