Sensitivity to Flg22 is modulated by ligand-induced degradation and de novo synthesis of the endogenous flagellin-receptor FLAGELLIN-SENSING2

Abstract

FLAGELLIN-SENSING2 (FLS2) is the plant cell surface receptor that perceives bacterial flagellin or flg22 peptide, initiates flg22-signaling responses, and contributes to bacterial growth restriction. Flg22 elicitation also leads to ligand-induced endocytosis and degradation of FLS2 within 1 h. Why plant cells remove this receptor precisely at the time during which its function is required remains mainly unknown. Here, we assessed in planta flg22-signaling competency in the context of ligand-induced degradation of endogenous FLS2 and chemical interference known to impede flg22-dependent internalization of FLS2 into endocytic vesicles. Within 1 h after an initial flg22 treatment, Arabidopsis (Arabidopsis thaliana) leaf tissue was unable to reelicit flg22 signaling in a ligand-, time-, and dose-dependent manner. These results indicate that flg22-induced degradation of endogenous FLS2 may serve to desensitize cells to the same stimulus (homologous desensitization), likely to prevent continuous signal output upon repetitive flg22 stimulation. In addition to impeding ligand-induced FLS2 degradation, pretreatment with the vesicular trafficking inhibitors Wortmannin or Tyrphostin A23 impaired flg22-elicited reactive oxygen species production that was partially independent of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1. Interestingly, these inhibitors did not affect flg22-induced mitogen-activated protein kinase phosphorylation, indicating the ability to utilize vesicular trafficking inhibitors to target different flg22-signaling responses. For Tyrphostin A23, reduced flg22-induced reactive oxygen species could be separated from the defect in FLS2 degradation. At later times (>2 h) after the initial flg22 elicitation, recovery of FLS2 protein levels positively correlated with resensitization to flg22, indicating that flg22-induced new synthesis of FLS2 may prepare cells for a new round of monitoring the environment for flg22.

Figure 1.

Flg22-induced degradation of endogenous FLS2 is ligand, dose, and time dependent. A and B, Time dependency and specificity in Ler seedlings. Ler wild-type and fls2-24 mutant seedlings were elicited in the presence (+) or absence (–) of 10 μm flg22 (A) or with 1 μm of indicated PAMPs (B) for indicated times. C, Dose and ligand dependency in Col-0 leaves. Col-0 wild-type leaf strips were elicited with (+) or without (–) indicated PAMP with indicated concentration for 0 or 60 min. For immunoblot analyses, total protein extracts were probed with αFLS2 (FLS2), αCalnexin (Caln), or αMPK6 (M6) antibodies. Probing with the latter two antibodies served as loading controls. Each experiment was done at least three times with similar results. AF, inactive flg22 (from A. tumefaciens); E, elf26.

Figure 2.

Ligand-induced desensitization to flg22 is PAMP and time dependent. A, Specificity of reelicitation of MAPK phosphorylation. Col-0 leaf strips were first elicited with 0.1 µm flg22 (PF) for 0, 10, or 60 min. Samples denoted 70* were reelicited at 60 min for 10 min with 0.1 µm of indicated PAMP; minus (–) denotes no elicitation at given time point. Total protein extracts were probed with αMPK6 (M6) or αP-p44/42 MAPK to assess MPK6 protein levels or phosphorylation of MPK6 (P-M6), MPK3 (P-M3), and an unknown MPK (P-M?). B–D, Specificity of ROS reelicitation. For first elicitation, Col-0 leaf discs were elicited with 0.1 µm of indicated PAMP at 0 min (white symbols), washed, and reelicited with indicated PAMP at 60 min (arrow; black symbols; n = 20 per treatment). All three ROS experiments in B to D were set-up in the same 96-well plate at the same time to allow direct comparison. E, Signaling competency between 20 and 60 min. All Col-0 leaf disc halves (n = 96) were elicited at 0 min with 0.1 μm active flg22 (white bar). After 15 min, all tissue was washed. Subsets of tissue (n = 16 per treatment) were then reelicited a second time at 20, 40, or 60 min with indicated PAMP. White bar represents ROS peak production 10 to 12 min after the first elicitation at 0 min (PF once). Black (PF/PF) or gray bars (PF/E) represent ROS peak production 10 to 12 min after reelicitation of tissue subsets for a second time at indicated times with active flg22 or elf26, respectively. To allow for direct comparison, all treatments in E were set-up in the same 96-well plate at the same time. Values are mean ± se, and means with different or the same letters are significantly different or not significantly different, respectively (two-tailed Student’s t test, P ≤ 0.0001). Each experiment was repeated more than three times with similar results. AF, inactive flg22; E, elf26; RLU, relative light unit.

Figure 3.

Desensitization of flg22-induced ROS production was dose dependent. A to D, For ROS production, Col-0 leaf disc halves were elicited for their first elicitation (white square) with the indicated concentration of flg22 at 0 min, washed, and reelicited at 60 min (arrow) with 0.1 μm flg22 (black square; n = 24 per treatment). To allow direct comparisons, all shown ROS experiments (A–D) were performed in the same 96-well plate at the same time. Values are mean ± se. This experiment was repeated more than three times with similar results. RLU, relative light units.

Figure 4.

Effects of chemical inhibitors on ligand-induced degradation of FLS2 and flg22 signaling. A and B, Pretreatment with 30 µm Wm. C and D, Pretreatment with 100 µm TyrA23. E and F, Pretreatment with 100 µm TyrA51. For immunoblot analyses (A, C, and E), Col-0 leaf strips were treated with (+) or without (–) chemical inhibitors and elicited with (+) or without (–) 1 µm flg22 for indicated times in minutes. Total protein extracts were probed with αFLS2, αP-p44/42 MAPK to assess FLS2 protein degradation, or flg22-induced phosphorylation of MPK6 (P-M6), MPK3 (P-M3), and an unknown MPK (P-M?). Individual MAPKs were identified by apparent mass. Immunoblots probed with αMPK6 (M6) confirmed MPK6 accumulation and served as loading control. For flg22-induced ROS production (B, D, and F), Col-0 leaf disc halves were treated in the presence (black squares) or absence (white squares) of chemical inhibitors and elicited with 1 µm flg22 at 0 min (n = 24 per treatment). Mock treated samples (x) were pretreated with either inhibitor and treated with DMSO instead of flg22 at 0 min (n = 24 per treatment). To allow for correct comparisons, ROS experiments shown in the same section were performed in the same 96-well plate at the same time. Values are mean ± se. Each experiment was done at least three times with similar results. T23, TyrA23; T51, TyrA51; RLU, relative light units.

Figure 5.

Cells remain flg22-signaling incompetent after pretreatment with vesicular trafficking inhibitors Wm and TyrA23 following an initial flg22 elicitation. A, Flg22-induced ROS reelicitation after pretreatment with 100 µm TyrA23. B, Flg22-induced ROS reelicitation after pretreatment with 30 µm Wm. For ROS production in A and B, Col-0 leaf disc halves were pretreated for 1 h with (circles) or without (square) chemical inhibitors, washed, elicited with 1 μm flg22 at 0 min (first elicitation, white symbols), and then reelicited at 60 min (arrow; second elicitation, black symbols) with 1 μm flg22. To allow for correct comparisons, ROS experiments shown in the same section (A or B) were performed in the same 96-well plate at the same time (n = 24 per treatment). Experiment was repeated at least three times with similar results. RLU, relative light units.

Figure 6.

TyrA23- and Wm-dependent inhibition of flg22-induced ROS is partially independent of BAK1. A, Flg22-induced ROS reelicitation in bak1-4 mutant plants. Col-0 or bak1-4 leaf disc halves were elicited with 1 μm flg22 at 0 min (first elicitation, white symbols) and then reelicited at 60 min (arrow; second elicitation, black symbols) with 1 μm flg22. B, Flg22-induced ROS production after pretreatment with (+) or without (–) 100 µm TyrA23 or 30 µm Wm in Col-0 or bak1-4. For ROS production, Col-0 or bak1-4 leaf disc halves were pretreated for 1 h with (+) or without (–) chemical inhibitors, washed, and then elicited once with 1 μm flg22 at 0 min. ROS peaks (10–12 min postelicitation) are shown in bar graph representation. To allow direct comparisons, ROS experiments shown in the same section were performed in the same 96-well plate at the same time (n = 24 per treatment). Values are mean ± se, and means with different or the same letters are significantly different or not significantly different, respectively (two-tailed Student’s t test, P ≤ 0.001). Experiments were repeated at least three times with similar results. RLU, relative light units.

Figure 7.

Ligand-induced degradation of FLS2 and attenuation of flg22-signaling responses is impaired by treatment with the protein synthesis inhibitor CHX. A, Flg22-induced expression levels of FLS2 mRNA. Col-0 leaf strips were elicited for 40 to 45 min with 1 μm flg22, washed, and incubated in the absence of flg22 until indicated times. Samples were processed for qRT-PCR using At2g28390 as the reference gene. For each time point, results of at least three independent experiments containing three biological and three technical repeats are shown. Values are mean ± se, and means with different or the same letters are significantly different or not significantly different, respectively (two-tailed Student’s t test, P ≤ 0.003). B, Effect of CHX on ligand-induced degradation of FLS2 and flg22-signaling responses. For immunoblot analyses, total protein extracts were probed with αFLS2, αP-p44/42 MAPK, or αMPK6 (M6) as in Figure 4. C, Effect of CHX on flg22-induced ROS production. Col-0 leaf disc halves (n = 24 per treatment) were cotreated in the presence (black squares) or absence (white squares) of 50 µm CHX and elicited with 1 µm flg22 at 0 min. Mock-treated samples (x) were treated with CHX and DMSO (instead of flg22) at 0 min (n = 24 per treatment). To allow for correct comparisons, ROS experiments shown in C were performed in the same 96-well plate at the same time. RLU, relative light units; P-M?, phosphorylated unknown MPK.

Figure 8.

FLS2 protein replenishment leads to resensitization to flg22 that is FLS2 and RbohD dependent. A, After initial ligand-induced degradation of FLS2, subsequent increased new FLS2 protein levels positively correlated with flg22-induced MAPK phosphorylation. After a first flg22 elicitation at 0 min for 45 to 50 min, Col-0 leaf strips were washed and incubated in the absence of flg22 until indicated times. For reelicitation, samples were reelicited with flg22 at indicated hours for 10 min. Immunoblot analysis of total protein extracts was done as in Figure 4. B to D, Resensitization of flg22-induced ROS production. All Col-0 leaf disc halves were elicited with flg22 at 0 h for 45 min (n = 60 per treatment). After a wash step, subsets of leaf disc halves (n = 20 per treatment) were reelicited at 1 (B), 3 (C), or 16 h (D). Arrows indicate times of second elicitation with flg22. ROS experiments shown in B, C, and D were set-up in the same 96-well plate to allow for direct comparison. E, Bar graph representation showing ROS peak (10–12 min) after first elicitation and second reelicitation with active flg22 at indicated hours (n = 10 per treatment). F, ROS resensitization is FLS2 dependent. G, ROS resensitization is RbohD dependent. For F and G, wild-type (Col-0), fls2, or rbohD leaf tissue was treated as in B to D at indicated times. For E to G, white bars represent ROS peak production 10 to 12 min after the first elicitation at 0 min (PF once), and black bars represent ROS peak production 10 to 12 min after reelicitation of tissue subsets for a second time at indicated times (PF/PF). ROS experiments shown in the same panel were set-up in the same 96-well plate. All experiments were elicited with 0.1 μm flg22 and done at least three times with similar results. Values are mean ± se. RLU, relative light unit; P-M?, phosphorylated unknown MPK; Caln, calnexin.