S-acylation of a non-secreted peptide controls plant immunity via secreted-peptide signal activation

Small peptides modulate multiple processes in plant cells, but their regulation by post-translational modification remains unclear. ROT4 (ROTUNDIFOLIA4) belongs to a family of Arabidopsis non-secreted small peptides, but knowledge on its molecular function and how it is regulated is limited. Here, we find that ROT4 is S-acylated in plant cells. S-acylation is an important form of protein lipidation, yet so far it has not been reported to regulate small peptides in plants. We show that this modification is essential for the plasma membrane association of ROT4. Overexpression of S-acylated ROT4 results in a dramatic increase in immune gene expression. S-acylation of ROT4 enhances its interaction with BSK5 (BRASSINOSTEROID-SIGNALING KINASE 5) to block the association between BSK5 and PEPR1 (PEP RECEPTOR1), a receptor kinase for secreted plant elicitor peptides (PEPs), thereby activating immune signaling. Phenotype analysis indicates that S-acylation is necessary for ROT4 functions in pathogen resistance, PEP response, and the regulation of development. Collectively, our work reveals an important role for S-acylation in the cross-talk of non-secreted and secreted peptide signaling in plant immunity.

(A) The positions of potential S-acylation sites on ROT4.The cysteine residues are indicated on the whole protein of ROT4.(B) Protein alignments of the conserved domain of Arabidopsis RTFL members.The sequences of the Arabidopsis RTFL proteins were aligned using ClustalW.The conserved domain is shown and the C42 residue in ROT4 is indicated.The phylogenetic tree was constructed using full-length protein sequences with a neighbor-joining method (bootstrap, 1000 replicates).

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Wenliang Li et al EMBO reports

Figure EV1 .
Figure EV1.Conservation of S-acylation sites in Arabidopsis RTFL members.

Figure EV2 .
Figure EV2.Contribution of S-acylation to the function of RTFL proteins.(A) Specificity verification of the cell fractionation assay.Free GFP (in cytosol fraction) or PAT12-GFP (in membrane fraction) was expressed in protoplasts.Total proteins (T) from protoplasts were divided into pellet (P) and soluble (S) fractions via ultra-centrifugation.The representative anti-GFP immunoblot from three biologically independent experiments is shown.(B, C) Effect of an R-to-C substitution on the subcellular localization of RTFL13.The WT or R41C version of GFP-RTFL13 was expressed in protoplasts.The representative immunoblotting results of cell fractionation from three biologically independent experiments are shown in (B).The representative GFP (green) and merged (with the bright field in gray and chloroplast auto-fluorescence in magenta) signals from three biologically independent experiments are shown in (C).Scale bars: 5 µm.(D-F) The phenotypes of RTFL13 and RTFL18 overexpressing plants.The expression level of RTFL13 or RTFL18 overexpressing lines was verified by RT-PCR (D).The RT-PCR data are representative of three biologically independent experiments.ACTIN1 was used as an internal control.The representative phenotypes of 2-weekold WT, vector control, and two independent lines of RTFL13 or RTFL18 overexpressing plants from three biologically independent experiments are shown (E, F).Source data are available online for this figure.

Figure EV3 .
Figure EV3.Effect of ROT4 S-acylation on its interaction with BSK5 in a BiFC assay.The indicated protein pairs were expressed in protoplasts for 24 h before confocal microscopy.YN or YC, the N or C fragment of YFP.The representative YFP (yellow) and Merged (with the bright field in gray and chloroplast autofluorescence in magenta) from three biologically independent experiments are shown.Scale bars: 5 µm.Source data are available online for this figure.

Figure EV4 .
Figure EV4.Effect of ROT4 overexpression and Pep1 treatment on the association of PEPR1 and phosphorylation defective BSK5.(A,B) Effect of ROT4 overexpression on the interaction between BSK5(S209A/T210A) and PEPR1.GFP or PEPR1-GFP was co-expressed with BSK5(WT)-Myc or BSK5(S209A/T210A)-Myc in protoplasts generated from wild-type or ROT4-overexpressing plants.ST: S209A/T210A.Co-IP was performed using anti-GFP Agarose and the representative immunoblots with anti-GFP or anti-Myc antibodies are shown in (A).The quantitative analysis of the BSK5-PEPR1 interaction from three biologically independent experiments is shown in (B).Immunoblot signals were quantified by ImageJ and the interaction intensity was calculated from relative signal ratios (pulldown/ input) of BSK5-Myc.The relative interaction intensity in the sample of BSK5(WT) in the WT cells was set to 1. (C, D) Effect of Pep1 on the interaction between BSK5(S209A/T210A) and PEPR1.GFP or PEPR1-GFP was co-expressed with BSK5(WT)-Myc or BSK5(S209A/T210A)-Myc in wild-type protoplasts with or without 1 μM of Pep1 treatment for 20 min.ST: S209A/T210A.Co-IP was performed using anti-GFP Agarose and the representative immunoblots with anti-GFP or anti-Myc antibodies are shown in (C).The quantitative analysis of the BSK5-PEPR1 interaction from three biologically independent experiments is shown in (D).Immunoblot signals were quantified by ImageJ and the interaction intensity was calculated from relative signal ratios (pulldown/input) of BSK5-Myc.The relative interaction intensity in the sample of BSK5(WT) under a control condition was set to 1. Data information: In (B, D), data are presented as mean ± SD; significance analysis was performed using one-way ANOVA followed by Tukey's multiple comparison tests (P < 0.05).Source data are available online for this figure.

Figure EV5 .
Figure EV5.Effect of ROT4 overexpression on development in the PEPR1 mutant background.(A) Verification of the transgenic plants by quantitative RT-PCR.ROT4 was overexpressed in the WT, pepr1-1, or pepr1-2 mutant background and the transgenic lines were verified by quantitative RT-PCR.ACTIN2 was used as an internal control.The relative expression level of ROT4 in the WT plants was set to 1.The quantitative RT-PCR data are from triplicated technological repeats, three biologically independent experiments showed similar patterns.(B) Developmental phenotypes of the indicated plants.Non-transgenic WT and PEPR1 mutants are included as controls.The 3-week-old plants were photographed.The representative image of plant phenotypes from three biologically independent experiments is shown.Data information: In (A), data are presented as mean ± SD; significance analysis was performed using one-way ANOVA followed by Tukey's multiple comparison tests (P < 0.05).Source data are available online for this figure.