Stem-cell-triggered immunity through CLV3p–FLS2 signalling

Stem cells in the shoot apical meristem (SAM) of plants are the selfrenewable reservoir for leaf, stem and flower organogenesis. In nature, disease-free plants can be regenerated from SAM despite infections elsewhere, which underlies a horticultural practice for decades. However, the molecular basis of the SAM immunity remains unclear. Here we show that the CLAVATA3 peptide (CLV3p), expressed and secreted from stem cells and functioning as a key regulator of stem-cell homeostasis in the SAM of Arabidopsis, can trigger immune signalling and pathogen resistance via the flagellin receptor kinase FLS2 (refs 5, 6). CLV3p– FLS2 signalling acts independently from the stem-cell signalling pathway mediated through CLV1 and CLV2 receptors, and is uncoupled from FLS2-mediated growth suppression. Endogenous CLV3p perception in the SAMby a pattern recognition receptor for bacterial flagellin, FLS2, breaks the previously defined self and nonself discrimination in innate immunity. The dual perception of CLV3p illustrates co-evolution of plant peptide and receptor kinase signalling for both development and immunity. The enhanced immunity in SAM or germ lines may represent a common strategy towards immortal fate in plants and animals. In both plants and animals, innate immunity is triggered through pattern recognition receptors (PRRs) in response tomicrobe-associated molecular patterns (MAMPs) to provide the first line of inducible defence. Plant receptor kinases represent the main functions of known plasmamembranePRRs forMAMPrecognition to distinguishnon-self from self. FLS2 is the first characterized plant leucine-rich-repeat (LRR) receptor kinase that perceives bacterial flagellin and launches convergent downstream signalling and defence pathways for potentially broad-spectrum pathogen resistance. The perception of bacterial flagellin is conserved in seed plants, and functional FLS2 orthologues are found from A. thaliana to rice. As FLS2 is expressed throughout the whole plant including the SAM (Supplementary Fig. 2), flagellin–FLS2 signalling could provide immune protection in different parts of the plant body after infections. While developing a plant expression system to screen for peptidemediated receptor-like kinase (RLK) signalling, we observed that the endogenously modified 12-amino-acid CLV3p (Supplementary Table 1) triggered similar responses as flg22 (the conserved 22-amino-acid peptide of bacterial flagellin) in mesophyll protoplasts. This findingwas unexpected becauseCLV3p is normally expressed, secreted and processed by the stem cells to control SAM maintenance via CLV1 and CLV2 receptors. Flg22 and CLV3p, but not DCLV3p lacking the last His residue, activated similar mitogen-activated protein kinase (MAPK) activities detected by the in-gel kinase assay (Fig. 1a and Supplementary Fig. 3). Highly purified CLV3p synthesized by different sources displayed the same activities, ruling out the contamination possibility. We sought to identify the CLV3p receptor in leaf cells by examiningMAPK activation in various receptor mutants. Neither the dominant clv1-1 mutant (Fig. 1a) nor the clv2-1 mutant (Supplementary Fig. 4) affected CLV3ptriggeredMAPK activation. Surprisingly, two independent fls2mutant alleles of Landberg erecta (Ler) and Columbia (Col-0), but not the efr-1 (the bacterial elongation factor EF-Tu receptor EFR) mutant, failed to support the activation of MAPKs by both flg22 and CLV3p (Fig. 1a and Supplementary Fig. 3). Complementation with the wildtype FLS2 gene (Fig. 1b), but not CLV1 (data not shown) in the fls2 mutant, confirmed that FLS2 could recognize both flg22 andCLV3p to mediate MAPK signalling. Importantly, CLV3p and flg22 activated similar early marker genes, including FRK1, WRKY29 and WRKY30 (Fig. 1c). FLS2 signalling requires the recruitment of the RLK BAK1 and the interaction between FLS2 and BAK1 represents the earliest event (within a minute) triggered by flg22 binding to FLS2 (refs 6, 14–16). Like flg22, CLV3p also induced the immediate interaction between FLS2 and BAK1 detected by reciprocal co-immunoprecipitation (Supplementary Fig. 5a, b). Consistently, CLV3p signalling monitored by MAPK activation was greatly diminished in the bak1-4 mutant (Supplementary Fig. 6). Notably, CLV3p pre-treatment could confer enhanced resistance to the pathogenic bacteria Pseudomonas syringaepv. tomatoDC3000 in anFLS2-dependentmanner (Fig. 1d). These comprehensive analyses strongly support our new finding that the conserved flagellin receptor FLS2 can recognize the stem-cell peptide CLV3p and trigger convergent innate immune signalling. Peptide titration experiments using the FLS2 and BAK1 coimmunoprecipitation assay showed that 1 nM flg22 was as potent as 1mM CLV3p (Fig. 2a and Supplementary Fig. 5c) required for SAM suppression. Intriguingly, flg22 and CLV3p peptides supported similar primary gene activation but distinct long-term growth effects