Endogenous plant signalling peptides regulate developmental and growth-related processes. Recent research indicates that some of these peptides are classified as phytocytokines as they have regulatory functions during plant immune responses. However, the mechanistic basis for phytocytokine-mediated immune modulation remains largely elusive. Here, we identify GOLVEN2 (GLV2) peptides as novel phytocytokines in Arabidopsis thaliana. By peptide application, precursor overexpression and loss-of-function studies we show that GLV2 enhances sensitivity of plants to elicitation with the bacterial flagellin epitope flg22. GLV2 is perceived by ROOT MERISTEM GROWTH FACTOR INSENSITIVE (RGI) receptors and RGI3 forms an flg22-induced complex with the flg22-receptor FLAGELLIN SENSING 2, suggesting that RGIs are part of activated pattern recognition receptor signalling platforms. GLV2 perception increases posttranscriptional FLS2 abundance and RGIs promote FLS2 protein accumulation. Thus, GLV-RGI signalling controls above ground plant immunity via a novel mechanism of phytocytokine activity.

A synthetic GLV2 reference peptide (DMDY[+80]NSANKKRP[+16]IHN; Y[+80] = tyrosine sulfation, P[+16] = proline hydroxylation) was used for PRM assay setup and optimization. PRM measurements were performed with a 50-min linear gradient on a Dionex Ultimate 3000 RSLCnano system coupled to a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in PRM and positive ionization mode. MS1 spectra (360–1300 m/z) were recorded at a resolution of 60,000 using an AGC target value of 3×106 and a MaxIT of 100 ms. Targeted MS2 spectra were acquired at 60,000 resolution with a fixed first mass of 100 m/z, after higher-energy collisional dissociation (HCD) with 26% NCE, an AGC target value of 1×106, a MaxIT of 118 ms and an isolation window of 1.3 m/z. Within a single PRM run 17 peptide precursors were targeted (the GLV2 target peptide in several charge states as well as 14 retention time reference peptides) without retention time scheduling. The cycle time was ~2.1 s, which lead to ~10 data points per chromatographic peak. PRM data analysis was carried out using the software tool Skyline (version 64-bit 20.2.0.286). Interferences and peak integration boundaries were reviewed manually, considering the ten most intense transitions (precursor-fragment ion pairs) of the GLV2 peptide. The total GLV2 peptide intensity was computed by summing up all transition intensities and normalizing them to the intensity of one retention time reference peptide (TFAHTESHISK), which was spiked into all samples at a constant concentration, and which eluted at a similar retention time (~22.8 min).

Preparations of apoplastic wash fluids were performed as previously described (Nakano et al., 2020). In brief, 4-5-week old soil-grown plants were detached from root tissue and submerged in apoplastic wash fluid buffer (5 mM NaC2H3O2-, 0.2 M CaCl2 ,pH 4.3) and vacuum infiltrated. Upon centrifugation, eluates were collected and subjected to chlorophenol extraction according to previous protocols (Ohyama et al., 2008). After precipitation, peptides were dissolved in H2O and analysed by targeted mass spectrometry using Parallel Reaction Monitoring (PRM).