Plant endogenous peptides are crucial for diverse aspects of plant physiology. Among them, C-TERMINALLY ENCODED PEPTIDEs (CEPs) have recently emerged as important regulators of plant growth and stress responses. CEPs are divided into two major subgroups: group I CEPs and the less studied group II CEPs. We recently demonstrated that group I CEPs coordinate cell surface receptor-mediated immunity with nitrogen status in Arabidopsis thaliana (hereafter Arabidopsis). To mount full group I CEP responsiveness, the three phylogenetically related CEP RECEPTOR 1 (CEPR1), CEPR2 and RECEPTOR-LIKE KINASE 7 (RLK7) are required. Here, we provide evidence that biotic stress induces expression of the group II CEP peptide CEP14. CEP14 and the related CEP13 and CEP15 trigger hallmark immune signalling outputs in a proline hydroxylation pattern-dependent manner in Arabidopsis. Genetic data indicate that group II CEP members contribute to cell surface receptor-mediated immunity against bacterial infection. We further show that group II CEP perception primarily depends on CEPR2. Our work provides new insights into CEP function during biotic stress and sheds new light on the complexity of sequence-divergent CEP signalling mediated by specific endogenous receptors.

Seven-week-old soil-grown Col-0, 35S::CEP4 #4, and 35S::CEP14 #1 Arabidopsis plants were sprayed with either PtoCOR⁻, a ddH2O solution containing 0.04% Silwet or left untreated. Afterwards, the plants were covered with lids and returned to short-day growth chambers. 24 h post-treatment, plant shoots were harvested in groups of three and submerged in 100 mL of apoplastic wash fluid (AWF) buffer (5 mM sodium acetate, 0.2 M calcium chloride, pH 4.3), freshly supplemented with protease inhibitor cocktail (Roche). The samples were vacuum infiltrated two times (2 min each), and the AWFs were collected by centrifugation at 800 × g for 20 minutes at 4°C. The eluates were subjected to chlorophenol extraction [45] with the modification of substituting methylmorpholine for N-ethylmorpholine. Peptidome enrichment was performed using PD-Miditrap G-10 size exclusion gravity columns (Cytiva, Amersham, UK). The samples were lyophilized and resuspended in 500 μL of ddH2O for subsequent analysis.

The CEP4 and CEP14 peptides were analyzed directly from AWFs by targeted MS using Parallel Reaction Monitoring (PRM). Prior to the analysis, synthetic CEP4 and CEP14 reference peptides were used to establish and optimize PRM assays, using a final concentration of 450 fmol (on column) per synthetic peptide. PRM data acquisition was performed with a 50-min linear gradient on a Dionex Ultimate 3000 RSLCnano system coupled to a Fusion Lumos mass spectrometer (Thermo Fisher Scientific). MS1 spectra (360–1300 m/z) were recorded at a resolution of 120,000 using an automatic gain control (AGC) target value of 4×105 and a maximum injection time (MaxIT) of 100 msec. Targeted MS2 spectra were acquired in the Orbitrap at 60,000 resolution using higher-energy collisional dissociation (HCD) with a 30% normalized collision energy (NCE), an AGC target value of 2×105, a MaxIT of 118 msec and a Quadrupole isolation window of 1.3 m/z. Within a single PRM run, maximally 20 peptide precursors were targeted with a retention time scheduling of 15 min. PRM data were analyzed using the Skyline software (version Skyline-daily 64-bit 24.1.1449; MacLean et al., 2010). From the synthetic peptide measurements the 10 most intense transitions (precursor-fragment ion pairs) per peptide precursor as well as retention time were determined. Endogenous CEP4 and CEP14 peptide intensities in AWF were manually evaluated und peptide intensities were calculated by summing of all transition intensities. If necessary, integration boundaries were manually adjusted and strongly interfered transitions were removed.