Introduction

Receptor kinase (RK) are structurally conserved proteins, featuring an extracellular domain, a single transmembrane region, and a cytoplasmic kinase domain. The cytoplasmic kinase domain consists of a protein kinase catalytic domain (PKC) and a juxtamembrane region, containing crucial phosphorylation sites on specific Ser and/or Thr residues essential for pattern-triggered immunity (PTI), the initial defense layer in plants (Mitra et al., 2015; Gada et al., 2022). PTI efficiently inhibits pathogen infection by activating defense responses, exemplified by the FLS2 signaling pathway (Li et al., 2016a; Zhai et al., 2021; Yu et al., 2023). Upon recognizing flg22, FLS2 interacts with the co-receptor Brassinosteroid-Insensitive 1-associated Kinase 1 (BAK1), initiating phosphorylation events through the activation of receptor-like cytoplasmic kinases (RLCKs) such as BOTRYTIS-INDUCED KINASE 1 (BIK1) to elicit downstream immune responses (Chinchilla et al., 2006; Li et al., 2016b; Majhi et al., 2021).

Protein phosphorylation, a vital posttranslational modification, plays a well-established and significant role. Research suggests that alterations in protein phosphorylation affect protein dynamics and subcellular trafficking (Palladino et al., 2022; Kontaxi et al., 2023). In plants, phosphorylation of proteins can coalesce into membrane microdomains, forming platforms for active protein function at the plasma membrane (PM) (Bücherl et al., 2017). For example, Xue et al. (2018) demonstrated that phosphorylation of the blue light receptor phot1 accelerates PM movement, enhancing interaction with AtRem1.3-mCherry, a sterol-rich lipid marker. This underscores protein phosphorylation’s crucial role in PM dynamics and membrane partitioning. Upon flg22 treatment, multiple FLS2 phosphorylation sites activate, with FLS2 Serine-938 phosphorylation playing a pivotal role in defense activation (Cao et al., 2013). While FLS2 Ser-938 is crucial for plant PTI, its impact on immunity through spatiotemporal dynamics remains unknown.

Membrane microdomains, dynamic structures rich in sterols and sphingolipids, are crucial in regulating plasma membrane (PM) protein behavior (Boutté et al., 2020). Specific proteins like Flotillins and Remorins can uniquely label these microdomains within living cells (Cui et al., 2018; Bücherl et al., 2017). Various stimuli can induce PM proteins to move into mobile or immobile microdomains, suggesting a connection between microdomains and signal transduction (Kim et al., 2018). For instance, Xing et al. (2022) demonstrated that sterol depletion significantly impacts the dynamics of flg22-activated FER–GFP, emphasizing microdomains’ role in the lateral mobility and dissociation of FER from the PM under flg22 treatment. However, the spatial coordination of FLS2 dynamics and signaling at the PM, along with their relationships with microdomains, remains poorly understood.

To investigate if FLS2 Ser-938 phosphorylation-mediated immune response is linked to its microdomain, we analyzed diffusion and dwell time of FLS2 phospho-dead and phospho-mimic mutants at the PM before and after flg22 treatments. Our results show that flg22-induced dynamic and dwell time changes are abolished in FLS2^S938A. Using FLIM-FRET and smPPI techniques, we discovered that FLS2 in different phosphorylation states exhibited distinct membrane microdomain distribution and endocytosis. Additionally, various phenotypic experiments demonstrated that the immune response of phosphorylated FLS2^S938D was similar to wild-type FLS2, while weakened in transgenic plants with dephosphorylated FLS2^S938A. These findings contribute to the theoretical foundation for studying plant immunity regulation through phosphorylation.

Results and Discussion

The Ser-938 phosphorylation site changed the spatiotemporal dynamics of flg22-induced FLS2 at the plasma membrane

Previous studies highlight the crucial role of membrane protein phosphorylation in fundamental cellular processes, including PM dynamics (Vitrac et al., 2019; Offringa et al., 2013). In vitro mass spectrometry (MS) identified multiple phosphorylation sites in FLS2. Genetic analysis further identified Ser-938 as a functionally important site for FLS2 in vivo (Cao et al., 2013). FLS2 Ser-938 mutations impact flg22-induced signaling, while BAK1 binding remains unaffected, thereby suggesting Ser-938 regulates other aspects of FLS2 activity (Cao et al., 2013). To unravel the immune response regulation mechanisms via FLS2 phosphorylation, we generated transgenic Arabidopsis plants expressing C-terminal GFP-fused FLS2, S938A, or S938D under the FLS2 native promoter in the fls2 mutant background (Figure S1A). Using VA-TIRFM with single-particle tracking (Figure 1A and 1B), we investigated the diffusion dynamics of FLS2 phospho-dead and phospho-mimic mutants, following previous reports (Geng et al., 2022). Upon flg22 treatment, FLS2^S938D-GFP spots exhibited longer motion trajectories, while FLS2^S938A-GFP spots were limited to shorter motion tracks (Figure 1C). The results indicate significant changes in the diffusion coefficients and motion ranges of FLS2/FLS2^S938D-GFP after flg22 treatment, whereas FLS2^S938A-GFP showed no significant differences (Figure 1D and 1E). Similar outcomes were observed using Uniform Manifold Approximation and Projection (UMAP) technology (Dorrity et al., 2020) and fluorescence recovery after photobleaching (FRAP) (Greig et al., 2021) (Figure 1F, 1G and S1B, F), supporting the essential role of the S938 phosphorylation site in flg22-induced lateral diffusion of FLS2 at the PM.

Figure 1.

Using VA-TIRFM, we analyzed FLS2 particle dwell time across various phosphorylation states. The results revealed that flg22 treatment significantly reduced the fluorescence trajectory of FLS2 molecules compared to the control (Figure 1H and S1C), indicating that Ser-938 phosphorylation influences flg22-induced dwell times of FLS2 at the PM. Subsequently, real-time dynamic analysis through the Kymograph technique provided spatiotemporal information via frame-by-frame tracking (Zhou et al., 2020). Compared to FLS2-GFP and FLS2^S938D-GFP, FLS2^S938A-GFP showed nearly linear fluctuations in fluorescence intensity under flg22 conditions, and the duration of fluorescence retention was essentially unchanged (Figure S1D). To further confirm this, we quantified FLS2 dwell times using exponential function fitting (Figure 1I and S1E). After flg22 treatment, FLS2^S938D-GFP dwell times significantly decreased, resembling those of FLS2-GFP. Although the FLS2^S938A-GFP plants, dwell times appeared to slightly decrease with flg22 treatment, the change was not significant. This aligns with previous findings that NRT1.1 phosphorylation affects dynamics and dwell time (Zhang et al., 2019). Additionally, numerous FLS2 exhibited short-lived dwell times, indicating abortive endocytic events associated with the endocytic pathway and signal transduction (Bertot et al., 2018). Therefore, these results underscore the impact of Ser-938 phosphorylation on the spatiotemporal dynamics of flg22-induced FLS2.

Ser-938 phosphorylation enhances recruitment of FLS2/BAK1 heterodimerization into AtRem1.3-associated microdomains

Proteins rarely act independently; they typically form multimers to enhance downstream signaling (Li et al., 2022). Previous studies have shown that inactive FLS2 mainly exists as monomers. Flg22 treatment induces FLS2 and BAK1 to heterodimerize at the PM, signifying flg22 as a ligand promoting this heterodimerization (Orosa et al., 2018). Therefore, we further investigated if Ser-938 phosphorylation affects FLS2/BAK1 heterodimerization. Tesseler segmentation, FRET-FLIM, and smPPI analyses revealed no impact of Ser-938 phosphorylation on FLS2/BAK1 heterodimerization (Figure 2A-C and S2). This aligns with the previous finding that flg22 acts as a molecular glue for FLS2 and BAK1 ectodomains (Sun et al., 2013), confirming the independence of FLS2/BAK1 heterodimerization from phosphorylation, with these events occurring sequentially.

Figure 2.

Membrane microdomains serve as pivotal platforms for protein regulation, impacting cellular signaling dynamics (Martinière et al., 2021). Studies reveal that specific plasma membrane (PM) proteins, responsive to developmental cues and environmental stimuli, exhibit dynamic movements within and outside these microdomains (Lee et al., 2019). For instance, treatment with the secreted peptides RAPID ALKALINIZATION FACTOR (RALF1 and RALF23) enhances the presence of FERONIA (FER) in membrane microdomains (Gronnier et al., 2022). Conversely, flg22-activated BSK1 translocates from membrane microdomains to non-membrane microdomains (Su et al., 2021). In a previous investigation, we demonstrated that flg22 induces FLS2 translocation from AtFlot1-negative to AtFlot1-positive nanodomains in the plasma membrane, implying a connection between FLS2 phosphorylation and membrane nanodomain distribution (Cui et al., 2018). To validate this, we assessed the association of FLS2/FLS2^S938D/FLS2^S938A with membrane microdomains, using AtRem1.3-associated microdomains as representatives (Huang et al., 2019). Employing SPT, FRET–FLIM, and Pearson correlation, we found increased correlation coefficients between FLS2-GFP/FLS2^S938D-GFP and AtRem1.3-mCherry during flg22 treatment compared to the control (Figure 2D-2F and Movies 1-3). Significantly, flg22 treatment did not increase colocalization levels of FLS2^S938A-GFP and AtRem1.3, indicating the necessity of phosphorylation (Figure 2G-2I). These findings suggest that the phosphorylation state of FLS2 at Ser-938 influences its aggregation into AtRem1.3-associated microdomains, providing an efficient mechanism for triggering the immune response.

Ser-938 phosphorylation maintains FLS2 protein homeostasis via flg22-induced endocytosis

The PM protein endocytosis is crucial for regulating intercellular signal transduction in response to environmental stimuli. Notably, Thr 867 mutation, a potential phosphorylation site on FLS2, results in impaired flg22-induced endocytosis, underscoring the significance of phosphorylation in FLS2 endocytosis (Robatzek et al., 2006). As mentioned earlier, FLS2 phosphomimetic mutants showed a reduced dwell time (Figures 1 H and 1I), implying a probable connection between FLS2 dwell time and endocytosis. Results found that FLS2/FLS2^S938D/FLS2^S938A-GFP accumulated in BFA compartments labeled with FM4-64, indicating that various Ser-938 phosphorylation states of FLS2 undergo BFA-dependent constitutive endocytosis (Figure 3A, S3A).

Figure 3.

Next, we investigated the impact of Ser-938 on flg22-induced FLS2 internalization. Results revealed increased endocytic vesicles for FLS2 during flg22 treatment, aligning with previous studies (Leslie et al., 2017; Loiseau et al., 2017). Employing confocal laser-scanning microscopy (CLSM) during 10μM flg22 treatment, we tracked FLS2 endocytosis and quantified vesicle numbers over time (Figure 3B). It is evident that both FLS2 and FLS2^S938D vesicles appeared 15 min after-flg22 treatment, significantly increasing thereafter (Figure 3C). Notably, only a few vesicles were detected in FLS2^S938A-GFP, indicating Ser-938 phosphorylation’s impact on flg22-induced FLS2 endocytosis. Additionally, fluorescence correlation spectroscopy (FCS) (Chen et al., 2009) monitored molecular density changes at the PM before and after flg22 treatment (Figure S3F). Figure 3D shows that both FLS2-GFP and FLS2^S938D-GFP densities significantly decreased after flg22 treatment, while FLS2^S938A-GFP exhibited minimal changes, indicating Ser-938 phosphorylation affects FLS2 internalization. Western blotting confirmed that Ser-938 phosphorylation influences FLS2 degradation after flg22 treatment (Figure 3E), consistent with single-molecule analysis findings. Therefore, our results strongly support the notion that Ser-938 phosphorylation expedited FLS2 internalization, potentially regulating its immune response capacity.

Ser-938 Phosphorylation affects FLS2-mediated responses

PTI plays a pivotal role in host defense against pathogenic infections (Lorrai et al., 2021; Ma et al., 2022). Previous studies demonstrated that FLS2 perception of flg22 initiates a complex signaling network with multiple parallel branches, including calcium burst, mitogen-activated protein kinases (MAPKs) activation, callose deposition, and seedling growth inhibition (Baral et al., 2015; Marcec et al., 2021; Huang et al., 2023). Our focus was to investigate the significance of Ser-938 phosphorylation in flg22-induced plant immunity. Figure 4A-F illustrates diverse immune responses in FLS2 and FLS2^S938D plants following flg22 treatment. These responses encompass calcium burst activation, MAPKs cascade reaction, callose deposition, hypocotyl growth inhibition, and activation of immune-responsive genes. In contrast, FLS2^S938A (Figure S4A-D) exhibited limited immune responses, underscoring the importance of Ser-938 phosphorylation for FLS2-mediated PTI responses.

Figure 4.

In summary, our study confirmed that FLS2 phosphorylation regulated PAMP-triggered plant immunity by influencing spatiotemporal dynamics at the PM (Figure 4 G and H). Following flg22 treatment, activated FLS2 undergoes hetero-oligomerization and phosphorylation sequentially. Crucially, phosphorylation at the Ser-938 site promotes FLS2 recruitment into AtRem1.3-associated microdomains and endocytosis. These results provided new insights into the phosphorylation-regulated dynamics of plant immunity, thereby providing a reference for future studies on signal transduction in intracellular complex nanostructures.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (32030010, 91954202, 32370740, 32000483, 32170689), the Fundamental Research Funds for the Central Universities (NO.QNTD202301), National Key Research and Development Program of China (2022YFF0712500, 2022YFD2200603), Beijing Nova Program (20230484251), 5·5 Engineering Research & Innovation Team Project of Beijing Forestry University (BLRC2023C06) and the program of Introducing Talents of Discipline to Universities (111 project, B13007).

Author contributions

J.L., Y.C. and X.L. designed the research; Y.C., H.Q., C.X. and J.Y. performed the research; C.X., P.L. and M.Y. contributed new reagents/analytic tools; X.Z., H.Q. and B.S. analyzed the data; and Y.C., H.Q. and J.L. wrote the paper.

Declaration of interests

The authors declare no competing interests.

STAR Methods

Plant Materials and Construction

Mutants and transgenic lines used in all experiments were in the Arabadopsis thaliana Colombia-0 (Col-0) background. To generate the transgenic plants, specific constructs were PCR-amplified and cloned into the vector pCAMBIA2300. Plasmids were introduced into the mutant plants by Agrobacterium-mediated transformation. Dual-color lines expressing FLS2^S938A-GFP and FLS2^S938D-GFP with REM1.3-mCherry were generated by hybridization.

Drug Treatments

All chemicals were obtained from Sigma-Aldrich, and dissolved in 100% DMSO to yield stock solutions at the following concentrations: BFA (50 mM in DMSO, 50 µM in working solution), CHX (50 mM in DMSO, 50 µM in working solution), and FM4-64 (5 mM in DMSO, 5 µM in working solution). The flg22 of flagellin peptides was synthesized by Shanghai GL Biochem Company, and was used in a concentration of 10 µM in double-distilled H2O. Arabidopsis seedlings were treated in 1/2 MS growth liquid medium with added hormone or drug.

Quantitative Reverse-transcription PCR

Total RNA was extracted using the Plant Kit (Tiangen), and then reverse transcribed into cDNA with FastQuant RT Kit (Tiangen). Next, qPCR was performed using TiangenSurperRealPreMix Plus (SYBR Geen). The primers used were as follows: WRKY33 (At At2g38470) 5′-GAAACAAATGGTGGGAATGG-3′ and 5′-TGTCGTGTGATGCTCTCTCC-3′; CYP81 (At At2g23190) 5′-AAATGGAGAGAGCAACACAATG-3′ and 5′-ATCGCCCATTCCAATGTTAC-3′; FRK1 (AtAt2g19190) 5′-TATATGGACACCGCGTATAGTG-3′and 5′-ATAAAACTTTGCGTTAGGGTCG-3′.

Aniline Blue Staining

To detect callose deposition, aniline blue staining was performed as described. Arabidopsis thaliana leaves were completely de-colored by the destaining solution (3 mL ethyl alcohol and 1 ml glacial acetic acid), rinsed in water and 50% ethanol, and then stained in 150 mM KH₂PO₄ (pH 9.5) plus 0.01% aniline blue for 2 h. Samples were mounted in 25% glycerol, and then observed under a microscope that was equipped with a Leica DM2500 UV lamp.

Confocal Laser Scanning Microscopy and Image Analysis

Confocal microscopy was done with a TCS SP5 Confocal Microscope fitted with a 63X water-immersion objective. GFP and FM4-64 were assayed using 488-nm and 514-nm wavelengths (multitrack mode). The fluorescence emissions were respectively detected with spectral detector set LP 560–640 (FM4-64) and BP 520–555 (GFP). Image analysis was performed with Leica TCS SP5 software and quantified using the ImageJ software bundle (NIH).

VA-TIRFM and Single-Particle Fluorescence Image Analysis

The dynamics of FLS2 phospho-dead and phospho-mimic mutants were recorded using VA-TIRFM. This was done using an inverted microscope (IX-71, Olympus) equipped with a total internal reflective fluorescence illuminator (model no. IX2-RFAEVA-2; Olympus) and a 1003 oil-immersion objective (numerical aperture = 1.45). To track GFP-labeled proteins at the PM, living leaf epidermal cells of 6-day-old seedlings were observed under VA-TIRFM. To visualize GFP or mCherry fluorescent proteins, appropriate corresponding laser excitation (473-nm or 561-nm) was used and emission fluorescence was obtained with filters (BA510IF for GFP; HQ525/50 for mCherry). A digital EMCCD camera (Andor Technology, ANDOR iXon DV8897D-CS0-VP, Belfast, UK) was used to acquire the fluorescent signals, which were stored directly on computers and then analyzed with Image J software. Images of single particles were acquired with 100-ms exposure time.

TIRF-SIM imaging and the colocalization analysis

The SIM images were taken by a 60 × NA 1.49 objective on a structured illumination microscopy (SIM) platform (DeltaVision OMX SR) with a sCMOS camera (Camera pixel size, 6.5 μm). The light source for TIRF-SIM included diode laser at 488 nm and 568 nm with pixel sizes (μm) of 0.0794 and 0.0794 (Barbieri et al., 2021).

For the dual-color imaging, FLS2/FLS2^S938A/FLS2^S938D-GFP (488 nm/30.0%) and AtRem1.3-mCherry (561 nm/30.0%) were excited sequentially. The exposure time of the camera was set at 50 ms throughout single-particle imaging. The time interval for time-lapse imaging was 100 ms, the total time was 2s, and the total time points were 21s. The Imaris intensity correlation analysis plugin was used to calculate the co-localization ratio.

Ca²⁺ Flux Measurements in Arabidopsis Leaves

Net Ca²⁺ fluxes in Arabidopsis leaf cells were measured using the non-invasive micro-test technique (NMT), as described previously (Zhong et al., 2023). First, a small incision was made in the leaves of fourteen-day-old seedling. Then it was fixed at the bottom of 35 mm petri dish, and incubated in the test buffer (pH =6.0, 0.1 mmol L⁻¹ KCl/CaCl₂/MgCl₂, 0.2 mmol L⁻¹ Na₂SO₄, 0.3 mmol L⁻¹ MES, 0.5 mmol L⁻¹ NaCl) Ca²⁺ concentration in the leaf cells was measured at 0.2 Hz near and 30 µm away from the cells. Each plant was measured once, and then use 1/2 MS (CK) or 10 μM flg22 treated the leaf and measured Ca²⁺flux again. The Ca²⁺ flux was calculated as described (Jiao et al., 2022).

Analysis of Root Growth

Arabidopsis seedlings were treated with different conditions, and imaging was performed by scanning the root systems at 500 dpi (Canon EOS 600D). ImageJ was used to analyze the root growth parameters. Three biological replicates were performed.

Fcs

FCS was performed in point-scanning mode on a Leica TCS SP5 FCS microscope equipped with a 488-nm argon laser, an Avalanche photodiode, and an in-house coupled correlator. After acquiring images on the PM of a cell in transmitted light mode, the diffusion of protein molecules into and out of the focal volume transformed the local concentration of fluorophores, leading to spontaneous fluctuation in the fluorescence intensity. Finally, the protein density was calculated on the basis of the protocol described previously.

Fret flim

FRET-FLIM analysis was performed using an inverted Olympus FV1200 microscope equipped with a Picoquant picoHarp300 controller. The excitation at 488 nm was implemented by a picosecond pulsed diode laser at a reduplication rate of 40 MHz, by way of a water immersion objective (603, numerical aperture [NA] 1.2). The emitted light passed through a 520/35 nm bandpass filter and was detected by an MPD SPAD detector. Data were collected and performed using the SymphoTime 64 software (PicoQuant).

Western Blot

Total proteins were extracted from 10-day-old seedlings of the acidic phosphomimic mutants FLS2^S938A-GFP and FLS2^S938D-GFP and transgenic FLS2-GFP lines under different conditions. Proteins were extracted using buffer E [includes 1.5125 g Tris-HCl (pH 8.8), 1.2409 g Na₂S₂O₅, 11.1 ml glycerine, 1 g SDS, and 5 mM DTT]. The proteins in PM fractions were obtained using the Invent Minute kit. Proteins were separated by 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blotted with anti-GFP antibody (Sigma-Aldrich) at a 1:4000 dilution.

Supplemental data

Supplemental Figure S1. Different Ser-938 phosphorylation states lead to specific spatiotemporal dynamics of FLS2.

Supplemental Figure S2. Different Ser-938 phosphorylation states do not affect the hetero-oligomerization of FLS2/BAK1.

Supplemental Figure S3. Effects of Ser-938 phosphorylation on the endocytosis of FLS2.

Supplemental Figure S4. Ser-938 phosphorylation affects flg22-induced PTI responses.

Supplemental Movies 1-3. TIRF-SIM movies of FLS2 (WT, A, D) and AtRem1.3-mCherry.