FERONIA regulates FLS2 plasma membrane nanoscale dynamics to modulate plant immune signaling

ABSTRACTCell surface receptors survey and relay information to ensure the development and survival of multicellular organisms. In the model plant Arabidopsis thaliana, the Catharanthus roseus RLK1-like receptor kinase FERONIA (FER) regulates myriad of biological processes to coordinate development, growth and responses to the environment. We recently showed that FER positively regulates immune signaling by controlling the ligand-induced complex formation between the leucine-rich repeat receptor kinase (LRR-RK) FLAGELLIN SENSING 2 (FLS2) and its co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3). In this context, FER function is inhibited by binding of its peptide ligand RAPID ALKALINIZATION FACTOR 23 (RALF23). However, the mechanisms by which FER regulates FLS2-BAK1 complex formation remain unclear. Here, we show that FER-dependent regulation of immune signaling is independent of its kinase activity, indicating that FER rather plays a structural role. FER has been proposed to bind directly to the plant cell wall, but we found that a FER mutant unable to bind pectin is still functional in regulating immune signaling. Instead, FER- and cell wall-associated LEUCINE RICH REPEAT-EXTENSIN proteins are required for this regulation. Using high-resolution live-imaging and single-particle tracking, we observed that FER regulates FLS2 plasma membrane nanoscale dynamics, which may explain its role in controlling ligand-induced FLS2-BAK1 association. We propose that FER acts as an anchoring point connecting cell wall and plasma membrane nano-environments to enable the nucleation of pre-formed receptor/co-receptor complexes at the cell surface.

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A conserved regulatory module regulates receptor kinase signaling in immunity and development

10.1101/2021.01.19.427293 ◽

2021 ◽

Author(s):

Thomas A. DeFalco ◽

Pauline Anne ◽

Sean R. James ◽

Andrew Willoughby ◽

Oliver Johanndrees ◽

...

Keyword(s):

Cell Surface ◽

Cellular Responses ◽

Receptor Signaling ◽

Receptor Kinase ◽

Surface Receptors ◽

Receptor Complexes ◽

Regulatory Circuit ◽

Regulatory Module ◽

Molecular Patterns ◽

Receptor Kinase Signaling

ABSTRACTLigand recognition by cell-surface receptors underlies development and immunity in both animals and plants. Modulating receptor signaling is critical for appropriate cellular responses but the mechanisms ensuring this are poorly understood. Here, we show that signaling by plant receptors for pathogen-associated molecular patterns (PAMPs) in immunity and CLAVATA3/EMBRYO SURROUNDING REGION-RELATED peptides (CLEp) in development employ a similar regulatory module. In the absence of ligand, signaling is dampened through association with specific type-2C protein phosphatases (PP2Cs). Upon activation, PAMP and CLEp receptors phosphorylate divergent cytosolic kinases, which, in turn, phosphorylate the phosphatases, thereby promoting their release from the receptor complexes. Our work reveals a regulatory circuit shared between immune and developmental receptor signaling, which may have broader important implications for plant receptor kinase-mediated signaling in general.

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The cell wall regulates dynamics and size of plasma-membrane nanodomains inArabidopsis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1819077116 ◽

2019 ◽

Vol 116 (26) ◽

pp. 12857-12862 ◽

Cited By ~ 18

Author(s):

J. F. McKenna ◽

D. J. Rolfe ◽

S. E. D. Webb ◽

A. F. Tolmie ◽

S. W. Botchway ◽

...

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Confocal Microscopy ◽

Particle Tracking ◽

Single Particle ◽

Cluster Size ◽

Particle Dynamics ◽

Single Particle Tracking ◽

Signaling Cascade ◽

Plant Plasma Membrane

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

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alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

The Journal of Cell Biology ◽

10.1083/jcb.82.3.614 ◽

1979 ◽

Vol 82 (3) ◽

pp. 614-625 ◽

Cited By ~ 140

Author(s):

M C Willingham ◽

F R Maxfield ◽

I H Pastan

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Coated Vesicles ◽

The Other ◽

Con A ◽

Coated Pits ◽

Cultured Fibroblasts ◽

Receptor Complexes ◽

Transmission Electron ◽

Epidermal Growth

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.

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Experimental Dissection of the Cellular Machinery Involved in Receptor Mediated Endocytosis and Translocation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010009909x ◽

1981 ◽

Vol 39 ◽

pp. 528-531

Author(s):

J.L. Salisbury

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Regulatory Protein ◽

Fold Increase ◽

Coated Pits ◽

Surface Distribution ◽

Receptor Mediated Endocytosis ◽

Calcium Dependent ◽

Receptor Complexes ◽

Human Lymphoblastoid Cell

The cultured human lymphoblastoid cell line WiL2 is a model system of choice for studies on receptor mediated endocytosis (RME). These cells display antigen receptor immunoglobulin of the IgM class (rIgM) as integral plasma membrane proteins which are present in diffuse cell surface distribution in unstimulated cells. Initially, rIgM occurs over uncoated regions of the plasma membrane. Crosslinking rIgM with multivalent antibody (ligand) results in the entry of ferritin-labelled ligand-rIgM complexes into the RME pathway (Figure 1). Stimulation of RME by ligand challenge results in an approximately three-fold increase in cell surface area displaying clathrin coats on the cytoplasmic face of the membrane. The newly formed coated pits are located directly beneath ferritin-labelled ligand-receptor complexes and their appearance is sensitive to the calmodulin directed drug trifluoperazine dihydrochloride (TFP). Calmodulin is a calcium dependent regulatory protein which recognizes local transient fluxes of cytoplasmic Ca+2 and activates a wide variety of enzymes and other protein systems. In addition, antibodies raised against calf brain calmodulin were used in indirect immunofluorescence studies.

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The Arabidopsis Leucine-Rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1

The Plant Cell ◽

10.1105/tpc.17.00376 ◽

2017 ◽

Vol 29 (9) ◽

pp. 2285-2303 ◽

Cited By ~ 37

Author(s):

Julia Imkampe ◽

Thierry Halter ◽

Shuhua Huang ◽

Sarina Schulze ◽

Sara Mazzotta ◽

...

Keyword(s):

Complex Formation ◽

Receptor Complex ◽

Receptor Kinase ◽

Leucine Rich Repeat

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A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin.

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4825 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4825-4833 ◽

Cited By ~ 86

Author(s):

C F Lu ◽

J Kurjan ◽

P N Lipke

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Plasma Membrane ◽

Cell Surface ◽

Soluble Form ◽

Gpi Anchor ◽

Glycosyl Phosphatidylinositol ◽

Experimental Support ◽

Cell Biol ◽

A Cell

Saccharomyces cerevisiae alpha-agglutinin is a cell wall-anchored adhesion glycoprotein. The previously identified 140-kDa form, which contains a glycosyl-phosphatidylinositol (GPI) anchor (D. Wojciechowicz, C.-F. Lu, J. Kurjan, and P. N. Lipke, Mol. Cell. Biol. 13:2554-2563, 1993), and additional forms of 80, 150, 250 to 300, and > 300 kDa had the properties of intermediates in a transport and cell wall anchorage pathway. N glycosylation and additional modifications resulted in successive increases in size during transport. The 150- and 250- to 300-kDa forms were membrane associated and are likely to be intermediates between the 140-kDa form and a cell surface GPI-anchored form of > 300 kDa. A soluble form of > 300 kDa that lacked the GPI anchor had properties of a periplasmic intermediate between the plasma membrane form and the > 300-kDa cell wall-anchored form. These results constitute experimental support for the hypothesis that GPI anchors act to localize alpha-agglutinin to the plasma membrane and that cell wall anchorage involves release from the GPI anchor to produce a periplasmic intermediate followed by linkage to the cell wall.

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TMK-based cell surface auxin signaling activates cell wall acidification in Arabidopsis

10.21203/rs.3.rs-203621/v1 ◽

2021 ◽

Author(s):

Zhenbiao Yang ◽

Wenwei Lin ◽

Wenxin Tang ◽

Koji Takahashi ◽

Hong Ren ◽

...

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Cell Surface ◽

Cell Expansion ◽

Auxin Signaling ◽

Molecular Evidence ◽

Signaling Proteins ◽

Acid Growth ◽

Cell Wall Acidification ◽

Growth Hypothesis

Abstract The phytohormone auxin controls a myriad of processes in plants, at least in part through its regulation of cell expansion. The "acid growth hypothesis" has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism underlying auxin-induced cell wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane (PM) H+-ATPase that pumps protons into the apoplast, yet how auxin activates its phosphorylation remains elusive. Here, we show that the transmembrane kinase (TMK) auxin signaling proteins interact with PM H+-ATPases and activate their phosphorylation to promote cell wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced TMK's interaction with H+-ATPase on the plasma membrane within 1-2 minutes as well as TMK-dependent phosphorylation of the penultimate Thr residue. Genetic, biochemical, and molecular evidence demonstrates that TMKs are required for auxin-induced PM H+-ATPase activation, apoplastic acidification, and cell expansion. Thus, our findings reveal a crucial connection between auxin and PM H+-ATPase activation in regulating apoplastic pH changes and cell expansion via TMK-based cell surface auxin signaling.

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LRR-extensins of vegetative tissues are a functionally conserved family of RALF1 receptors interacting with the receptor kinase FERONIA

10.1101/783266 ◽

2019 ◽

Author(s):

Aline Herger ◽

Shibu Gupta ◽

Gabor Kadler ◽

Christina Maria Franck ◽

Aurélien Boisson-Dernier ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Cell Wall ◽

Plasma Membrane ◽

Cell Growth ◽

Mechanical Stability ◽

Root Hairs ◽

Cell Wall Integrity ◽

Receptor Kinase ◽

Growth Processes ◽

Evolutionary Diversification

AbstractPlant cell growth requires the coordinated expansion of the protoplast and the cell wall that confers mechanical stability to the cell. An elaborate system of cell wall integrity sensors monitors cell wall structures and conveys information on cell wall composition and growth factors to the cell. LRR-extensins (LRXs) are cell wall-attached extracellular regulators of cell wall formation and high-affinity binding sites for RALF (rapid alkalinization factor) peptide hormones that trigger diverse physiological processes related to cell growth. RALF peptides are also perceived by receptors at the plasma membrane and LRX4 of Arabidopsis thaliana has been shown to also interact with one of these receptors, FERONIA (FER). Here, we demonstrate that several LRXs, including the main LRX protein of root hairs, LRX1, interact with FER and RALF1 to coordinate growth processes. Membrane association of LRXs correlate with binding to FER, indicating that LRXs represent a physical link between intra- and extracellular compartments via interaction with membrane-localized proteins. Finally, despite evolutionary diversification of the LRR domains of various LRX proteins, many of them are functionally still overlapping, indicative of LRX proteins being central players in regulatory processes that are conserved in very different cell types.Author SummaryCell growth in plants requires the coordinated enlargement of the cell and the surrounding cell wall, which is ascertained by an elaborate system of cell wall integrity sensors, proteins involved in the exchange of information between the cell and the cell wall. In Arabidopsis thaliana, LRR-extensins (LRXs) are localized in the cell wall and are binding RALF peptides, hormones that regulate cell growth-related processes. LRX4 also binds the plasma membrane-localized receptor kinase FERONIA (FER), establishing a link between the cell and the cell wall. It is not clear, however, whether the different LRXs of Arabidopsis have similar functions and how they interact with their binding partners. Here, we demonstrate that interaction with FER and RALFs requires the LRR domain of LRXs and several but not all LRXs can bind these proteins. This explains the observation that mutations in several of the LRXs induce phenotypes comparable to a fer mutant, establishing that LRX-FER interaction is important for proper cell growth. Some LRXs, however, appear to influence cell growth processes in different ways, which remain to be identified.

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