Clathrin and Membrane Microdomains Cooperatively Regulate RbohD Dynamics and Activity in Arabidopsis    

Abstract

Arabidopsis thaliana respiratory burst oxidase homolog D (RbohD) functions as an essential regulator of reactive oxygen species (ROS). However, our understanding of the regulation of RbohD remains limited. By variable-angle total internal reflection fluorescence microscopy, we demonstrate that green fluorescent protein (GFP)-RbohD organizes into dynamic spots at the plasma membrane. These RbohD spots have heterogeneous diffusion coefficients and oligomerization states, as measured by photobleaching techniques. Stimulation with ionomycin and calyculin A, which activate the ROS-producing enzymatic activity of RbohD, increases the diffusion and oligomerization of RbohD. Abscisic acid and flg22 treatments also increase the diffusion coefficient and clustering of GFP-RbohD. Single-particle analysis in clathrin heavy chain2 mutants and a Flotillin1 artificial microRNA line demonstrated that clathrin-and microdomain-dependent endocytic pathways cooperatively regulate RbohD dynamics. Under salt stress, GFP-RbohD assembles into clusters and then internalizes into the cytoplasm. Dual-color fluorescence cross-correlation spectroscopy analysis further showed that salt stress stimulates RbohD endocytosis via membrane microdomains. We demonstrate that microdomain-associated RbohD spots diffuse at the membrane with high heterogeneity, and these dynamics closely relate to RbohD activity. Our results provide insight into the regulation of RbohD activity by clustering and endocytosis, which facilitate the activation of redox signaling pathways.

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

“…These findings demonstrate that the ROS burst is most likely conferred by plasma membrane-localized FLS2, in line with the activation of RbohD via an upstream calcium influx, calcium-dependent protein kinases, and BOTRYTIS INDUCED KINASE (BIK1) (29,30). Although our results are in disagreement with other findings showing opposing outcomes of either strongly reduced or prolonged oxidative bursts upon inhibition of flg22-induced FLS2 degradation or endocytosis, respectively (31, 32), we cannot exclude the possibility of additional effects from the chemical approaches used in these studies or from the activities of clathrin-mediated endocytosis regulating RbohD localization (33). Knockout of CHC1 did not alter the flg22-triggered ROS burst (Fig.…”

Plasma Membrane Microdomains Are Essential for Rac1-RbohB/H-Mediated Immunity in Rice

“…Proteome analyses have identified Rbohs in DRMs of tobacco, Arabidopsis and rice (Morel et al, 2006;Kierszniowska et al, 2009;Fujiwara et al, 2009), and ROS production by Rbohs in pollen tube tips is dependent on PM microdomains in Picea meyeri (Liu et al, 2009). Recent reports also showed that the dynamics of AtRbohD are regulated by PM microdomains (Hao et al, 2014). At-RbohD has been shown to play an important role in innate immunity in Arabidopsis (Kadota et al, 2015) and belongs to same clade with Os-RbohB and Os-RbohH (Supplemental Figure 11A).…”

WRKY Transcription Factors Phosphorylated by MAPK Regulate a Plant Immune NADPH Oxidase in Nicotiana benthamiana

“…The flg22-PTI ROS burst peaked 30 to 40 min after treatment and returned to nearly basal levels within 60 min ( Figure 6A), in that activated RBOHs on the plasma membrane might have been rapidly inactivated or degraded. The dynamics of RBOHs after elicitation were observed recently in living Arabidopsis cells: Heterogeneously distributed RBOHD on the plasma membrane appeared to internalize into the cytoplasm, leading to endocytosis after ROS generation (Hao et al, 2014). In tobacco cultured cells, RBOHD-GFP seems to exist on the plasma membrane and at the periphery of Golgi cisternae, and elicitation with oomycete protein cryptogein activates transcriptional upregulation of RBOHD and distribution of RBOHD-GFP to the plasma membrane from the Golgi pool; a protein synthesis inhibitor accelerates the decrease in RBOHD-GFP fluorescence in intracellular compartments (Noirot et al, 2014).…”

“…GFP-tagged WRKY8-chromatin complex directly phosphorylates and activates Arabidopsis RBOHD to induce PTI ROS burst (Kadota et al, 2014;Li et al, 2014). Confocal images of At-RBOHD promoter:GFP-At-RBOHD transgenic seedlings showed that GFP-At-RBOHD proteins accumulated in untreated Arabidopsis leaves (Hao et al, 2014), indicating that some amount of RBOHD exists on the plasma membrane for rapid reaction mediated by BIK1 upon the PAMP sensing. ROS bursts induced by INF1 treatment (Supplemental Figure 13; Chaparro-Garcia et al, 2011) and R3a/AVR3a coexpression ( Figures 6B and 6C) are sustained and require the transcriptional activation of RBOHB via the MAPK-WRKY pathway ( Figure 6C).…”