Danger-associated peptide signaling in Arabidopsis requires clathrin

Abstract

The Arabidopsis thaliana endogenous elicitor peptides (AtPeps) are released into the apoplast after cellular damage caused by pathogens or wounding to induce innate immunity by direct binding to the membrane-localized leucine-rich repeat receptor kinases, PEP RECEPTOR1 (PEPR1) and PEPR2. Although the PEPR-mediated signaling components and responses have been studied extensively, the contributions of the subcellular localization and dynamics of the active PEPRs remain largely unknown. We used live-cell imaging of the fluorescently labeled and bioactive pep1 to visualize the intracellular behavior of the PEPRs in the Arabidopsis root meristem. We found that AtPep1 decorated the plasma membrane (PM) in a receptor-dependent manner and cointernalized with PEPRs. Trafficking of the AtPep1-PEPR1 complexes to the vacuole required neither the trans-Golgi network/early endosome (TGN/EE)-localized vacuolar H + -ATPase activity nor the function of the brefeldin A-sensitive ADP-ribosylation factor-guanine exchange factors (ARF-GEFs). In addition, AtPep1 and different TGN/EE markers colocalized only rarely, implying that the intracellular route of this receptor-ligand pair is largely independent of the TGN/EE. Inducible overexpression of the Arabidopsis clathrin coat disassembly factor, Auxilin2, which inhibits clathrin-mediated endocytosis (CME), impaired the AtPep1-PEPR1 internalization and compromised AtPep1-mediated responses. Our results show that clathrin function at the PM is required to induce plant defense responses, likely through CME of cell surface-located signaling components.Arabidopsis | clathrin | endogenous peptides | PEPR | endocytosis

Clathrin in plant defense signaling and execution

“…Until recently, there was only very limited insight into what happens to PRKs in plant cells upon the arrival of the signal: that is, after specific ligand binding resulting in an intracellular kinase domain activation and signaling initiation. Two side-byside reports in PNAS (7,8) show that three different PRKs [PEP receptor 1 (PEPR1), EF-TU RECEPTOR (EFR), and FLAGELLIN SENSING 2 (FLS2)], involved in biotic defense interactions activated by three different ligands, are all removed from the cell surface via clathrin-mediated endocytosis (CME). An interacting coreceptor, BRI1-ASSOCIATED KINASE 1 (BAK1), is necessary for the internalization of all these activated receptors.…”

“…In a pathway seemingly bypassing the TGN/EE, all three internalized ligand-bound receptors converge on the same ARA7/RabF2b marker-positive late-endosomal prevacuolar compartment (PVC), which is identical to the multivesicular body (MVB) and destined finally for the vacuole (7,8) (Fig. 1).…”

“…1). Interestingly, temporal dynamics for endocytotic internalization of the steroid receptor BRI1 were speedier when compared with pathogen-related pattern recognition ligandbound FLS2 or PEPR1 receptors, but the final destination was the same, indicating a possible existence of the late-endosomal compartment as a general depot for internalized activated RKs in plant cells (6)(7)(8).…”

“…Substantial differences with respect to the importance of CME for the initiation of early signaling pathways were observed between FLS2 versus PEPR RKs. Although flg22-induced/FLS2 RK-dependent transient activation of reactive oxygen species production (via RBOH NADPH oxidase/NOX) and MAPK signaling was not inhibited concomitantly with clathrin endocytosis inhibition (7), in the case of Pep1-induced/PEPR-dependent activation, MAPK signaling output was strongly inhibited by endocytosis down-regulation (8). As in the case of FLS2, activated BRI1 internalization is not necessary for the signaling; in contrast, retention of activated brassinosteroid-bound BRI1 at the PM stimulated signal output (6).…”

“…Interestingly, only clathrin heavy chain 2 (CHC2)-dependent endocytosis affects the internalization of the activated receptors; down-regulation of CHC1 has no effect (7,8). FLS2-dependent flg22-induced stomata closure is impaired in chc2 mutants, whereas ABA-imposed closure still can take place.…”