Spatio-Temporal Cellular Dynamics of the Arabidopsis Flagellin Receptor Reveal Activation Status-Dependent Endosomal Sorting

Abstract

The activity of surface receptors is location specific, dependent upon the dynamic membrane trafficking network and receptor-mediated endocytosis (RME). Therefore, the spatio-temporal dynamics of RME are critical to receptor function. The plasma membrane receptor flagellin sensing2 (FLS2) confers immunity against bacterial infection through perception of flagellin (flg22). Following elicitation, FLS2 is internalized into vesicles. To resolve FLS2 trafficking, we exploited quantitative confocal imaging for colocalization studies and chemical interference. FLS2 localizes to bona fide endosomes via two distinct endocytic trafficking routes depending on its activation status. FLS2 receptors constitutively recycle in a Brefeldin A (BFA)-sensitive manner, while flg22-activated receptors traffic via ARA7/Rab F2b- and ARA6/Rab F1-positive endosomes insensitive to BFA. FLS2 endocytosis required a functional Rab5 GTPase pathway as revealed by dominant-negative ARA7/Rab F2b. Flg22-induced FLS2 endosomal numbers were increased by Concanamycin A treatment but reduced by Wortmannin, indicating that activated FLS2 receptors are targeted to late endosomes. RME inhibitors Tyrphostin A23 and Endosidin 1 altered but did not block induced FLS2 endocytosis. Additional inhibitor studies imply the involvement of the actin-myosin system in FLS2 internalization and trafficking. Altogether, we report a dynamic pattern of subcellular trafficking for FLS2 and reveal a defined framework for ligand-dependent endocytosis of this receptor.

Danger-associated peptide signaling in Arabidopsis requires clathrin

“…Because TGN/EE is the first compartment that gathers endocytosed cargos (26), high colabeling was expected between the TGN/EE markers and TAMRA-pep1, as previously observed for the BRI1 receptor-ligand complexes (8). Surprisingly, and similar to FLS2 (7,27), the colocalization of TAMRA-pep1 with all tested TGN/EE markers, including clathrin (28) and VHA-a1, was low, in contrast to the MVB markers, for which high colocalization values were obtained. Possibly, the AtPep1-PEPR complexes either are internalized directly to MVBs bypassing the TGN/EE or are transported to the MVBs via a V-ATPasenegative subpopulation of the TGN.…”

“…TAMRA-pep1 and PEPR-GFP were simultaneously internalized and transported to the vacuole, most probably as a mechanism to desensitize cells after the AtPep1 stimulation, as reported for other ligands (8,25). Interestingly, the temporal dynamics of the AtPep1-PEPR complexes differed from those of BR-BRI1, which internalize very rapidly (8), but were similar to those of the FLS2 receptor (7). Thus, diverse signaling responses require endocytosis with different dynamics and, notably, pattern recognition receptors follow ligand-induced endosomal trafficking with similar dynamics.…”

“…Despite the ever-growing number of plant signaling peptides, their intracellular dynamics have not been reported to date. PEPRs are structurally similar to the plasma membrane (PM)-localized receptor kinases FLAGELLIN SENSING2 (FLS2) and BRASSINOSTEROID INSENSITIVE1 (BRI1), which undergo endocytosis both independently and dependently of their ligands, the bacterial peptide flagellin 22 (flg22) and the brassinosteroid (BR) hormone, respectively (7,8).…”

ENDOSOMAL RAB EFFECTOR WITH PX-DOMAIN, an Interacting Partner of RAB5 GTPases, Regulates Membrane Trafficking to Protein Storage Vacuoles in Arabidopsis

“…ARA6 acts in the endosome-to-plasma membrane trafficking pathway (Ebine et al, 2011) and may also be involved in a vacuolar trafficking pathway (Bottanelli et al, 2011). ARA7 and RHA1, in contrast, redundantly regulate endocytic and vacuolar trafficking pathways (Sohn et al, 2003;Kotzer et al, 2004;Dhonukshe et al, 2006;Ebine et al, 2011Ebine et al, , 2014Beck et al, 2012). The unique organization of endosomal trafficking in plants is also demonstrated by the observation that the trans-Golgi network (TGN) acts as an early endosome in plants (Dettmer et al, 2006;Lam et al, 2007;Viotti et al, 2010), from which multivesicular endosomes are proposed to be directly generated by maturation (Niemes et al, 2010;Scheuring et al, 2011).…”

LYM2-dependent chitin perception limits molecular flux via plasmodesmata

“…S2). After our observation that FLS2 mediates flg22-triggered closure of PD, we carefully examined leaves of transgenic lines expressing FLS2-GFP (22) and found unevenly distributed signals in the plasma membrane. Similar to LYM2-mCit, bright domains of fluorescence corresponded with aniline bluestained PD-associated callose (Fig.…”