RABA Members Act in Distinct Steps of Subcellular Trafficking of the FLAGELLIN SENSING2 Receptor

Abstract

Cell surface proteins play critical roles in the perception of environmental stimuli at the plasma membrane (PM) and ensuing signal transduction. Intracellular localization of such proteins must be strictly regulated, which requires elaborate integration of exocytic and endocytic trafficking pathways. Subcellular localization of Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a receptor that recognizes bacterial flagellin, also depends on membrane trafficking. However, our understanding about the mechanisms involved is still limited. In this study, we visualized ligand-induced endocytosis of FLS2 using green fluorescent protein (GFP)-tagged FLS2 expressed in Nicotiana benthamiana. Upon treatment with the flg22 peptide, internalized FLS2-GFP from the PM was transported to a compartment with properties intermediate between the trans-Golgi network (TGN) and the multivesicular endosome. This compartment gradually discarded the TGN characteristics as it continued along the trafficking pathway. We further found that FLS2 endocytosis involves distinct RABA/RAB11 subgroups at different steps. Moreover, we demonstrated that transport of de novo-synthesized FLS2 to the PM also involves a distinct RABA/RAB11 subgroup. Our results demonstrate the complex regulatory system for properly localizing FLS2 and functional differentiation in RABA members in endoand exocytosis.

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.…”

“…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. In agreement, the transport of TAMRA-pep1 to the vacuole, although delayed, was not blocked by the inhibitor of the V-ATPase activity, ConcA (13), similarly to the boron transporter BOR1 at high boron concentrations (29) and the FLS2 receptor (7,27). Intriguingly, ARA7 colocalized with the VHA-a1 in the TGN/EE, thus marking the TGN/EE subdomains that will mature into MVBs (30).…”

Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases

“…1 A and C). As reported previously (15), N. benthamiana-expressed FLS2-GFP localized to the plasma membrane and was internalized when triggered with flg22 or wild type (WT) Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) extracts ( Fig.…”

“…To support our findings, we tested whether PEPR1 and EFR traffic along the same endocytic pathway as revealed for FLS2 (5,15), and pairwise coexpressed in N. benthamiana leaves PEPR1-YFP, FLS2-GFP, and EFR-GFP with markers of the Golgi apparatus (MEMB12), the trans-Golgi network (TGN; Vha-A1), and two partially overlapping populations of endosomes (ARA7/ RabF2b and ARA6/RabF1). Upon cognate ligand elicitation, vesicles positive for each receptor showed colocalization with mCherry-ARA7/RabF2b-labeled and ARA6/RabF1-RFP-labeled endosomes, whereas no colocalization was observed with MEMB12-mCherry or Vha-A1-RFP (Fig.…”

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

“…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). The RAB11 group (also known as RABA in Arabidopsis), which is localized to TGNrelated secretory compartments and is involved in cell plate formation and tip growth (Preuss et al, 2004;Chow et al, 2008;Szumlanski and Nielsen, 2009;Kang et al, 2011;Feraru et al, 2012;Asaoka et al, 2013;Berson et al, 2014;Kirchhelle et al, 2016), is also implicated in endosomal/vacuolar trafficking (Bottanelli et al, 2011;Choi et al, 2013). These lines of evidence indicate that endosomal trafficking in plant cells operates in a different manner from that in non-plant organisms.…”