Abstract
Plant signalling peptides are typically released from larger precursors by proteolytic cleavage to regulate plant growth, development and stress responses. Recent studies reported the characterization of a divergent family of Brassicaceae-specific peptides, SERINE RICH ENDOGENOUS PEPTIDES (SCOOPs), and their perception by the leucine-rich repeat receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2). Here, we reveal that the SCOOP family is highly expanded, containing at least 50 members in the Columbia-0 reference Arabidopsis thaliana genome. Notably, perception of these peptides is strictly MIK2-dependent. How bioactive SCOOP peptides are produced, and to what extent their perception is responsible for the multiple physiological roles associated with MIK2 are currently unclear. Using N-terminomics, we validate the N-terminal cleavage site of representative PROSCOOPs. The cleavage sites are determined by conserved motifs upstream of the minimal SCOOP bioactive epitope. We identified subtilases necessary and sufficient to process PROSCOOP peptides at conserved cleavage motifs. Mutation of these subtilases, or their recognition motifs, suppressed PROSCOOP cleavage and associated overexpression phenotypes. Furthermore, we show that higher-order mutants of these subtilases show phenotypes reminiscent of mik2 null mutant plants, consistent with impaired PROSCOOP biogenesis, and demonstrating biological relevance of SCOOP perception by MIK2. Together, this work provides insights into the molecular mechanisms underlying the functions of the recently identified SCOOP peptides and their receptor MIK2.
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The authors declare that the data supporting the findings of this study are available within the manuscript and its supplementary files. Source data are provided with this paper.
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Acknowledgements
We thank past and present members of the Zipfel laboratory for helpful discussions and comments. B. Brandt and P. Köster are particularly thanked for their assistance with the cloning of the PROSCOOP cleavage constructs. P. Pimprikar is also thanked for her assistance with generation of the CRISPR mutants. We acknowledge generous funding to study plant immune signalling by the Gatsby Charitable Foundation (C.Z.), the Biotechnology and Biological Research Council (BB/P012574/1) (C.Z.), the European Research Council under the European Union’s Horizon 2020 research and innovation programme no. 773153 (project ‘IMMUNO-PEPTALK’) (C.Z.) and programme no. 724321 (project ‘Sense2SurviveSalt’) (C.T.), the University of Zurich (C.Z.) and the Swiss National Science Foundation grants no. 31003A_182625 (C.Z.) and 310030_184769 (C.S.R.).
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H.Y., J.R. and C.Z. conceived and designed the experiments. H.Y., J.R., X.K., J.S., S.A., G.S.A., E.S., M.-C.G., N.G.-B, L.T.V.C. and K.W.B. generated materials, performed experiments and/or analysed the data. P.R., C.S.R., A. Stintzi, C.T., J.P.R., F.L.H.M., A. Schaller, J.R. and C.Z. supervised the project. H.Y., J.R. and C.Z. wrote the manuscript with input from all authors.
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Extended data
Extended Data Fig. 1 Sequence alignment of Arabidopsis PROSCOOPs.
Signal peptide, variable regions containing the predicted cleavage motifs RxLx/RxxL/VWD, and the active epitope containing the conserved motif SxS are indicated by coloured boxes.
Extended Data Fig. 2 All PROSCOOPs identified by RNA-Seq are up-regulated upon elicitor treatment.
Heat map showing log2(FC) expression levels of PROSCOOPs identified by RNA-Seq in response to a range of elicitors (data obtained from27).
Extended Data Fig. 3 Divergent SCOOPs-induced responses.
(a) Integrated ROS production over 40 min in leaf disks collected from 4-week-old Arabidopsis mik2-1 plants induced in the absence (Mock) or presence of 1 μM 13-mer SCOOPs peptides (n = 4) using 100 nM flg22 and elf18 as control. (b) Cytoplasmic Ca2+ influx measured in mik2-1AEQ seedlings induced in the absence (Mock) or presence of 1 μM 13-mer SCOOPs peptides (n = 4) using 100 nM flg22 and elf18 as control. (c) Integrated ROS production over 40 min in leaf disks collected from 4-week-old Arabidopsis plants induced in the absence (Mock) or presence of 1 μM 13/15-mer SCOOP8 peptides (n = 8) using 100 nM elf18 as control. (d) Integrated ROS production over 40 min in leaf disks collected from 4-week-old Arabidopsis mik2-1 plants induced in the absence (Mock) or presence of 15-mer SCOOPs (n = 6) using 100 nM elf18 as control. (e) Integrated ROS production over 40 min in leaf disks collected from 4-week-old Arabidopsis bak1-5 bkk1 plants relative to Col-0 induced in the absence (Mock) or presence of 1 μM 13-mer SCOOPs peptides (n = 4) using 100 nM flg22 and elf18 as control. (a-e) Data represents the mean ± SD; P-values indicate significance relative to the Mock in a Two-tailed T-test. All experiments were repeated and analysed three times with similar results.
Extended Data Fig. 4 Similarity relationships inside the PROSCOOP family and summary of SCOOPs activity in Col-0.
CLANS clustering based on all-against-all pairwise sequence similarities resulted in 7 groups (highlighted with different colours) and 6 singletons. P-values lower than 1.E-2 and 1.E-5 are represented by grey and black edges respectively. Coloured asterisk indicates effects of SCOOP peptides.
Extended Data Fig. 5 Analysis of SBT3 subgroup.
(a) SBT3.3/3.4/3.5 are co-regulated with MIK2 in response to different stresses. Heat map showing log2(FC) expression levels of SBTs in response to stresses (data obtained from Genevestigator). (b) Transcriptional regulation of Arabidopsis SBT genes by elicitors. Heat map showing log2(FC) expression levels of SBT genes in response to a range of elicitors (data obtained from27). (c) Phylogeny of Arabidopsis SBT3 subgroup. Phylogeny of the full-length amino acid sequences of SBT3 was inferred using the online T-coffee server (https://tcoffee.crg.eu/apps/tcoffee/do:mcoffee).
Extended Data Fig. 6 Cleavage analysis of PROSCOOP12 and PROSCOOP20 by different SBTs.
Related to Fig. 2b, indicating flg22-induced SBT3.5-mediated PROSCOOP12 cleavage.
Extended Data Fig. 7 The diagram of TMT-labelling coupled MS.
The working flow of TMT-labelling coupled MS to identify SBT3.5-mediated 6xHA-PROSCOOP12-GFP cleavage sites and SBT3.6-mediated 6xHA-PROSCOOP20-GFP cleavage sites in Nicotiana benthamiana.
Extended Data Fig. 8 Mass spectrometry of TMT labelling peptide.
(a) TMT-GSGAGPVR peptide was detected by N-terminal labelling-coupled mass spectrometry with three independent experiments. (b) TMT-DLKIGASGSNSG peptide was detected by N-terminal labelling-coupled mass spectrometry with three independent experiments. (c) TMT-TLLRDLKIGASGSNSG peptide was detected by N-terminal labelling-coupled mass spectrometry with only one time from three repeats.
Extended Data Fig. 9 CRISPR deletes the majority of SBT3.3/3.4/3.5 and SBT3.6/3.7/3.8/3.9/3.10 genomic region.
(a) Schematic view of the large genomic deletion of clusters SBT3.3/3.4/3.5. The fusion protein may be transcribed, consisting of the first 331 amino acids of SBT3.5 with an out-of-frame fusion with SBT3.3 leading to an early stop. Primers F1/R1 and F2/R2 used for the genotyping and sequence (Supplementary Table 3). (b) Schematic view of the large genomic deletion of clusters SBT3.6/3.7/3.8/3.9/3.10. One fusion protein driven by the promoter SBT3.7 may be transcribed, consisting of 212 amino acid of SBT3.7, fused to 12 nonsense amino acids from SBT3.6 genomic region. Another fusion protein driven by the promoter SBT3.6 may be transcribed, consisting of 726 amino acids of SBT3.6, fused to 11 nonsense amino acids from SBT3.7 genomic region. Primers F3/R3 and F4/R4 used for the genotyping and sequence (Supplementary Table 3).
Extended Data Fig. 10 Phenotypic analysis of sbt higher-order mutants.
(a) Phenotype of 5-week-old plants. Bar = 1cm. (b) Dry weight of 4-week-old plants. One week after germination, plants were transferred to pots with soil watered from below in demineralized water. After 3 weeks the rosettes were cut, and dry weight was determined. Data represents the mean ± SD of three independent experiments. P-values indicate significance relative to Col-0 in a two-sided T-test. Number of individual larvae measured: Col-0 n = 191, mik2-1 n = 160 and sbt3octuple n = 143. (c) Integrated ROS production over 30 min in leaf disks collected from 4-week-old Arabidopsis plants induced by 100 nM flg22 application (n = 8). (d) Quantification of the root angle of 9-day-old seedlings grown in an upright position on MS agar medium (n = 15). (e) Cumulative Arabidopsis root vascular penetration by Fo5176 in WT (Col-0), mik2-1, and sbt3octuple plants at different days post transfer (dpt) to plate-containing spores (n = 60). Data represents the mean ± SD of three independent experiments. Statistical significance calculated via repeated measures two-way ANOVA with Tukey post-hoc test (p value ≤ 0.05 (genotype), p value ≤ 0.05 (time). P values are indicated in the graph with respect to WT at any dpt. (b-d) Data represents the mean ± SD; P-values indicate significance relative to Col-0 in a Two-tailed T-test. All experiments were repeated and analysed three times with similar results.
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Supplementary Tables 1–3.
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Source Data 1
Statistical source data for Figs 1–4 and Extended Data Figs. 3 and 10.
Source Data 2
Unprocessed western blots for Figs 2 and 3 and Extended Data Fig. 6.
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Yang, H., Kim, X., Skłenar, J. et al. Subtilase-mediated biogenesis of the expanded family of SERINE RICH ENDOGENOUS PEPTIDES. Nat. Plants 9, 2085–2094 (2023). https://doi.org/10.1038/s41477-023-01583-x
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DOI: https://doi.org/10.1038/s41477-023-01583-x
