Mutations in FLS2 Ser-938 dissect signaling activation in FLS2-mediated Arabidopsis immunity

Abstract

Flagellin-sensing 2 (FLS2) is a leucine-rich repeat/transmembrane domain/protein kinase (LRR-RLK) that is the plant receptor for bacterial flagellin or the flagellin-derived flg22 peptide. Previous work has shown that after flg22 binding, FLS2 releases BIK1 kinase and homologs and associates with BAK1 kinase, and that FLS2 kinase activity is critical for FLS2 function. However, the detailed mechanisms for activation of FLS2 signaling remain unclear. The present study initially identified multiple FLS2 in vitro phosphorylation sites and found that Serine-938 is important for FLS2 function in vivo. FLS2-mediated immune responses are abolished in transgenic plants expressing FLS2(S938A), while the acidic phosphomimic mutants FLS2(S938D) and FLS2(S938E) conferred responses similar to wild-type FLS2. FLS2-BAK1 association and FLS2-BIK1 disassociation after flg22 exposure still occur with FLS2(S938A), demonstrating that flg22-induced BIK1 release and BAK1 binding are not sufficient for FLS2 activity, and that Ser-938 controls other aspects of FLS2 activity. Purified BIK1 still phosphorylated purified FLS2(S938A) and FLS2(S938D) mutant kinase domains in vitro. Phosphorylation of BIK1 and homologs after flg22 exposure was disrupted in transgenic Arabidopsis thaliana plants expressing FLS2(S938A) or FLS2(D997A) (a kinase catalytic site mutant), but was normally induced in FLS2(S938D) plants. BIK1 association with FLS2 required a kinase-active FLS2, but FLS2-BAK1 association did not. Hence FLS2-BIK1 dissociation and FLS2-BAK1 association are not sufficient for FLS2-mediated defense activation, but the proposed FLS2 phosphorylation site Ser-938 and FLS2 kinase activity are needed both for overall defense activation and for appropriate flg22-stimulated phosphorylation of BIK1 and homologs.

Figure 1. Identification of Ser-938 as a candidate autophosphorylation site of FLS2 in vitro and in vivo.

A and B. Intact (A) and antarctic phosphatase-treated (B) intracellular domains of FLS2 (aa #840-1172) were analyzed by Mass Spectrometry (MS). M: predicted molecular weight; 1P: predicted peptide with one phosphate group; 2P: predicted peptide with two phosphate groups. C. Peptides containing phosphorylated amino acids, identified by mass spectrometry (see also Figure S1). D–F. Functional test of three serine sites identified by MS. Reactive oxygen species were measured in leaf discs from transgenic Arabidopsis fls2-101 plants for 30 min. after treatment with 1 µM flg22. Stable transgenic plants carried FLS2 serine mutant alleles as specified, with expression driven by native FLS2 promoter. Data shown are mean ± SE for four to six independent T1 plants per construct. RLU: relative luminescence units; wt: wild-type Col-0 FLS2; S938A: FLS2_S938A; other FLS2 alleles similarly labeled.

Figure 2. Ser-938 is critical for FLS2-mediated physiological responses.

Mutant S938A, S938D, or S938E forms of FLS2 or wild-type FLS2 (WT), as indicated, were expressed under control of FLS2 promoter in Arabidopsis Col-0 fls2-101. Kanamycin/hygromycin-resistant T2 transgenic plants (from T1 plants shown in Figure S2A) were examined after treatment with 1 µM flg22. A–D. Representative images of callose production in leaves 24 hr after flg22 treatment. E. MPK phosphorylation in seedlings before (−) or 15 min. after (+) flg22 treatment, revealed by immunoblot after SDS-PAGE using the anti-P44/P42 antibody. Lower gel reveals similar loading of total protein. F. Response to flg22 in seedling growth inhibition assays. Relative growth is seedling fresh weight after flg22 treatment, divided by average weight of untreated seedlings. G. Leaf populations of P. syringae pv. tomato strain DC3000 3 days after inoculation. Data are mean ±SE for three separate experiments each involving three replicates (total n = 9). * and ** indicate T-test significant difference compared with Col-0 at P<0.05 and P<0.01, respectively.

Figure 3. Interaction of BAK1 or BIK1 with variants of FLS2.

HA-tagged full-length FLS2 wild type (WT), S938A, S938D or D997A (as labeled), and cMyc-tagged full length BAK1 or BIK1, were coexpressed under control of 35S promoters in Arabidopsis fls2-101 protoplasts. Protoplasts were harvested prior to (−) or 15 min. after (+) treatment with 1 µM flg22. Coimmunoprecipitation was carried out using anti-cMyc antibody. Input blots are from SDS-PAGE of total protein extracts; each lane was loaded with equivalent volumes of total protoplasts. WB: antibody used to probe immunoblot. A. FLS2_WT, FLS2_S938A and FLS2_S938D interact with BAK1 upon flg22 treatment. B. BIK1 dissociates from FLS2_WT, FLS2_S938A and FLS2_S938D after flg22 treatment. C. FLS2-FLS2 association before and after flg22 exposure is not reduced when both FLS2 partners carry the FLS2_S938A mutation. FLS2-BAK1 interaction from the same experiment is shown as a control (all six lanes in C from same protoplast batch, gel, blot and immunodetection). D. FLS2_D997A interacts with BAK_D416A upon flg22 treatment. FLS2_D997A does not interact as well as FLS2_WT with BIK1_D202A before flg22 treatment, and flg22-elicited release of BIK1 is not detected. E. FLS2_S938D, and separately, FLS2_D997A, form FLS2-FLS2 associations before and after flg22 treatment.

Figure 4. Transphosphorylation of FLS2 kinase domain by BIK1.

Kinase domains of FLS2 (wild-type or mutant as specified), and full-length wild-type BIK1 were purified from E. coli as GST fusion proteins and used for in vitro kinase assays in the presence of [γ ³²P]-ATP. Upper panel: autoradiograph after SDS-PAGE; lower panel: replicate gel of same samples, stained with Coomassie brilliant blue. Left and right panels are from same experiment but are from two separate gels; gels were exposed together for same period.

Figure 5. Induced phosphorylation of BIK1 and its homologous proteins under flg22 treatment.

cMyc tagged BIK1, PBS1, PBL1, and PBL2 were transiently expressed (under control of 35S promoters) in protoplasts made from Arabidopsis fls2-101 lines stably transgenic for full-length FLS2_WT, FLS2_S938A, FLS2_S938D, and FLS2 _D997A (under control of FLS2 promoters; Figure S2A). Protoplasts were treated with (+) or without (−) 1 µM flg22 for 15 min prior to harvest. Total protein extracts were separated by SDS-PAGE and immunoblots were probed with anti-cMyc to detect protein size shift attributable to phosphorylation. Lower panel of each pair shows Ponceau S staining of same immunoblot to assess similarity of total protein levels.