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. 2015 Jan 21;167(3):1076–1086. doi: 10.1104/pp.114.250985

The Arabidopsis Transcription Factor BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 Is a Direct Substrate of MITOGEN-ACTIVATED PROTEIN KINASE6 and Regulates Immunity1

Sining Kang 1,2,3, Fan Yang 1,2,3, Lin Li 1,2,3, Huamin Chen 1,2,3, She Chen 1,2,3, Jie Zhang 1,2,3,*
PMCID: PMC4348755  PMID: 25609555

MAP kinase-mediated phosphorylation of a transcription factor regulates plant immunity.

Abstract

Pathogen-associated molecular patterns (PAMPs) are recognized by plant pattern recognition receptors to activate PAMP-triggered immunity (PTI). Mitogen-activated protein kinases (MAPKs), as well as other cytoplasmic kinases, integrate upstream immune signals and, in turn, dissect PTI signaling via different substrates to regulate defense responses. However, only a few direct substrates of these signaling kinases have been identified. Here, we show that PAMP perception enhances phosphorylation of BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1), a transcription factor involved in brassinosteroid (BR) signaling pathway, through pathogen-induced MAPKs in Arabidopsis (Arabidopsis thaliana). BES1 interacts with MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and is phosphorylated by MPK6. bes1 loss-of-function mutants display compromised resistance to bacterial pathogen Pseudomonas syringae pv tomato DC3000. BES1 S286A/S137A double mutation (BES1SSAA) impairs PAMP-induced phosphorylation and fails to restore bacterial resistance in bes1 mutant, indicating a positive role of BES1 phosphorylation in plant immunity. BES1 is phosphorylated by glycogen synthase kinase3 (GSK3)-like kinase BR-insensitive2 (BIN2), a negative regulator of BR signaling. BR perception inhibits BIN2 activity, allowing dephosphorylation of BES1 to regulate plant development. However, BES1SSAA does not affect BR-mediated plant growth, suggesting differential residue requirements for the modulation of BES1 phosphorylation in PTI and BR signaling. Our study identifies BES1 as a unique direct substrate of MPK6 in PTI signaling. This finding reveals MAPK-mediated BES1 phosphorylation as another BES1 modulation mechanism in plant cell signaling, in addition to GSK3-like kinase-mediated BES1 phosphorylation and F box protein-mediated BES1 degradation.

Plants are challenged with pathogenic microbes during their whole life cycle. Conserved molecular patterns derived from bacteria and fungi are recognized by plant cell surface-localized pattern recognition receptors (PRRs) to activate pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI; Schwessinger and Zipfel, 2008). Several receptor kinases such as flagellin sensing2 (FLS2) and elongation factor thermo unstable (EF-Tu) receptor have been identified as PRRs recognizing bacterial flagellin and EF-Tu, respectively (Gómez-Gómez and Boller, 2000; Chinchilla et al., 2006; Zipfel et al., 2006). PTI confers broad and durable resistance against microbial pathogens. However, PTI signal transduction mechanisms remain not well understood. A few important cytoplasmic kinases, including mitogen-activated protein kinases (MAPKs), calcium-dependent protein kinases, and receptor-like cytoplasmic kinases, have been identified to integrate and amplify immune signals from activated PRRs (Veronese et al., 2006; Pitzschke et al., 2009; Boudsocq et al., 2010; Lu et al., 2010; Zhang et al., 2010) and in turn regulate downstream defense responses. The direct substrates for PTI signaling cytoplasmic kinases in PTI remain not well characterized.

In FLS2-mediated signaling pathway, botrytis-induced kinase1 (BIK1) and AVRPPHB SUSCEPTIBLE1-like1 (PBL1) associate with FLS2 prior to flagellin perception (Lu et al., 2010; Zhang et al., 2010). Binding of flagellar N-terminal peptide flg22 to FLS2 triggers recruitment of BRASSINOSTEROID-INSENSITIVE1-associated receptor kinase1 (BAK1) to FLS2 (Gómez-Gómez et al., 1999; Chinchilla et al., 2006, 2007; Heese et al., 2007; Sun et al., 2013) and also induces BIK1 phosphorylation and its dissociation from FLS2 (Lu et al., 2010; Zhang et al., 2010). Plant U-box (PUB) proteins PUB12 and PUB13 ubiquitinate FLS2 for degradation to attenuate PTI (Lu et al., 2011). Most recently, Arabidopsis respiratory burst oxidase homolog D (AtrbohD) has been reported to act as a direct substrate of BIK1 and contribute to BIK1-regulated stomatal defense (Kadota et al., 2014; Li et al., 2014). Downstream of PRR activation, both MITOGEN-ACTIVATED PROTEIN KINASE KINASE4 (MKK4)/MKK5-MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3)/MPK6 cascades and MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE1 (MEKK1)-MKK1/MKK2-MPK4 cascades are activated (Pitzschke et al., 2009). Mitogen-activated protein kinase4 substrate1 (MKS1) is a direct substrate of MPK4 that cooperates with WRKY33 to regulate target gene expression (Andreasson et al., 2005). 1-Aminocyclopropane-1-carboxylic acid synthase ACS2 and ACS6 are substrates of MPK3/MPK6 and regulate ethylene production in response to pathogen invasion (Liu and Zhang, 2004; Han et al., 2010; Li et al., 2012). AtPHOS32 (for phosphorylated protein with an apparent molecular mass of 32 kDa), ethylene response factor104 (AtERF104), and VIRULENCE PROTEIN E2-interacting protein1 (VIP1) are the only identified substrates of MPK6 and/or MPK3 in PTI, whereas their biological roles in PTI remain to be further characterized (Peck et al., 2001; Djamei et al., 2007; Merkouropoulos et al., 2008; Bethke et al., 2009). Identification of additional MAPK substrates is critical for uncovering the molecular mechanism of PTI signaling dissection.

Immune signals integrated by PTI signaling kinases induce the transcriptional reprogramming of a large number of defense-related genes. Histone methylation is a key epigenetic mechanism that regulates chromatin structure and gene expression. Thus, histone-modifying enzymes and related transcription regulators are candidate substrates for PTI signaling kinases. Several histone methyltransferases have been reported to regulate plant resistance against bacterial and fungal pathogens (Alvarez-Venegas et al., 2007; Berr et al., 2010). However, whether histone demethylase (HDM) is involved in plant immunity remains uncertain. BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1) is a major transcription factor in brassinosteroid (BR)-mediated signaling pathway. BR regulates BES1 activity through phosphorylation and dephosphorylation to control BR-responsive gene expression (Yin et al., 2002, 2005). BES1 has also been reported to recruit HDM EARLY FLOWERING6 (ELF6) and RELATIVE OF ELF6 to regulate BR-responsive gene expression (Yu et al., 2008). In addition, BES1 also participates in strigolactone (SL) signaling via F box protein-mediated degradation to control shoot branching (Wang et al., 2013).

In this study, we uncover a unique function of BES1 in plant immunity. We demonstrate that perception of flg22, a bacterial PAMP, enhances phosphorylation of BES1. This enhanced phosphorylation is prevented by the expression of either a bacterial MAPK inhibitor, hypersensitive response and pathogenicity-dependent outer protein AI1 (HopAI1), or the MKK5K99M dominant negative mutant, indicating a MAPK-dependent BES1 phosphorylation triggered by PAMP perception. Further analyses prove that BES1 interacts with MPK6 and is phosphorylated by MPK6 in vitro. bes1 mutants are compromised in bacterial resistance against Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Furthermore, BES1 S286A/S137A double mutation (BES1SSAA) impairing PAMP-induced phosphorylation fails to restore bacterial resistance in bes1 mutant but does not affect BR-mediated plant growth. Taken together, these data demonstrate BES1 as a direct substrate of MPK6 in PTI signaling and indicate differential modulation of a common transcription factor targeted by distinct signaling proteins in hormone and immunity signaling in plants.

RESULTS

BES1 Contributes to Plant Immunity to P. syringae

To investigate the role of histone demethylation in plant immune regulation, we carried out a reverse genetic screen to identify HDMs as well as HDM-bound transcription factors involved in Arabidopsis (Arabidopsis thaliana) bacterial resistance. Transfer DNA insertion lines of HDMs and HDM-bound transcription factors were inoculated with P. syringae strain Pst DC3000, and then in planta bacterial growth was assessed in mutant plants. Transfer DNA insertion mutants bes1-1 (He et al., 2005) and bes1-2 (Supplemental Fig. S1A), which supported more bacterial growth of Pst DC3000 (Fig. 1A) and developed more severe disease symptoms than ecotype Columbia (Col-0) wild-type plants (Supplemental Fig. S1B), were isolated. Quantitative reverse transcription-PCR data showed that BES1 expression in bes1-1 and bes1-2 mutants is significantly decreased compared with that in wild-type plants (Supplemental Fig. S1C). To study whether BES1 regulates the expression of PTI-responsive genes, bes1 mutants were treated with flg22 and the induction of PTI-responsive genes was measured. A reduction in WRKY22 and FLG22-INDUCED RECEPTOR-LIKE KINASE1 (FRK1) induction by flg22 was detected in bes1 mutants compared with wild-type plants (Fig. 1, B and C). Compromised bacterial resistance and defensive gene induction in bes1 mutants indicate that BES1 positively regulates plant immunity against bacterial pathogens.

Figure 1.

Figure 1.

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BES1 contributes to plant immunity. A, Growth of Pst DC3000 in wild-type (WT; Col-0), bes1-1, and bes1-2 plants. Wild-type or mutant plants were infiltrated with 105 cfu mL–1 Pst DC3000, and leaf bacterial population was determined at the indicated times. The results shown are representative of three independent experiments. An asterisk indicates significant difference at P < 0.05. B, BES1 is required for full induction of WRKY22 by Flg22. C, BES1 is required for full induction of FRK1 by Flg22. Five-week-old wild-type or bes1 mutant plants were infiltrated with or without 500 nm flg22 for 3 h. Total RNA was extracted and used for reverse transcription. Real-time PCR was performed following standard protocols. Values are normalized to TUBLIN control and are presented as relative to the value of water-treated wild-type plants. Error bars indicate sd of three technical repeats. Asterisks indicate significant difference to the flg22-treated wild type at P < 0.01. Similar results were observed in three independent biological repeats.

Flagellin Perception Enhances BES1 Phosphorylation

BES1 activity is regulated by phosphorylation and dephosphorylation in BR signaling pathway. In the absence of BR, BES1 is phosphorylated by the upstream glycogen synthase kinase3 (GSK3)-like kinase BR-insensitive2 (BIN2) and retained in the cytoplasm (Yin et al., 2002, 2005; Ryu et al., 2010). Perception of BR triggers dephosphorylation of BES1 and results in its accumulation in the nucleus to direct expression of BR-responsive genes (Yin et al., 2002; Ryu et al., 2010). We then speculated a modulation of BES1 phosphorylation by PAMP perception. BES1 native promoter-driven BES1 tagged with a FLAG epitope (BES1::BES1-FLAG) transgenic plants were constructed and used to study BES1 phosphorylation in response to PAMP treatment (Supplemental Materials and Methods S1). In untreated plants, both faster-migrating and slower-migrating forms of BES1 can be detected, as previously reported (Yin et al., 2002; Albrecht et al., 2012). We found that flg22 treatment resulted in the appearance of an intermediate-migrating BES1 in BES1::BES1-FLAG transgenic plants (Fig. 2, A and B, asterisk indicated). The appearance of this intermediate-migrating BES1 was removed by the treatment of λ protein phosphatase (PPase), indicating an elevated phosphorylation of BES1 upon flg22 stimulation (Fig. 2A). Epibrassinolide (EpiBL) has been shown to trigger dephosphorylation of BES1 (Yin et al., 2002; Albrecht et al., 2012). We wondered whether flg22 is able to resume phosphorylation of dephosphorylated BES1 in epiBL-pretreated plants. Hence, BES1::BES1-FLAG plants were pretreated with epiBL to eliminate basal level of phosphorylated BES1 and then treated with flg22. The following flg22 treatment clearly resumed phosphorylation of BES1 (Fig. 2B). This result confirms an elevated BES1 phosphorylation triggered by PAMP perception in plants. In agreement with the results from BES1::BES1-FLAG transgenic plants, flg22 treatment resulted in a slower mobility shift of BES1 in Arabidopsis protoplasts transfected with 35S::BES1-FLAG when compared with water treatment (Fig. 2C). Similar to that in transgenic plants, the mobility shift was removed by treatment of λ PPase (Fig. 2C), indicating an elevated phosphorylation of transiently expressed BES1 upon flg22 stimulation in protoplast. In addition to flg22, Crab shell chitin and elf18 (an N-acetylated peptide comprising the first 18 amino acids of EF-Tu) treatments also induced phosphorylation of BES1 (Supplemental Fig. S2). Taken together, the above results proved an elevated phosphorylation of BES1 triggered by PAMP perception in plants.

Figure 2.

Figure 2.

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Flg22 enhances BES1 phosphorylation. A, Flg22 induces BES1 phosphorylation in plants. BES1::BES1-FLAG transgenic plants were treated with water or 1 μm flg22 for 5 min, and total protein extracted from leaves was treated with or without PPase as indicated. B, Flg22 induces BES1 rephosphorylation after BL pretreatment in plants. BES1::BES1-FLAG transgenic plants were pretreated with or without 1 μm epiBL as indicated for 1 h, followed by 1 μm flg22 treatment for 5 min. C, Flg22 induces BES1 phosphorylation in protoplasts. Col-0 protoplasts were transfected with 35S::BES1-FLAG, treated with or without 1 μm flg22. Total protein extracted from protoplasts was treated with or without PPase as indicated. Protein samples were separated by SDS-PAGE and subjected to anti-FLAG immunoblot. Ponceau S (PS) staining of the filter indicates loading of the protein.

BES1 Phosphorylation Induced by flg22 Occurs Downstream of MAPK Activation

PAMP perception triggers both activation of MAPK cascades and phosphorylation of BIK1/PBL1, which are likely to act in two branches to regulate downstream responses (Zhang and Zhou, 2010). To determine the causal relationship between BES1 phosphorylation and other early signaling events, we next examined BES1 phosphorylation induced by flg22 in bik1/pbl1 double mutant, because BIK1 and PBL1 function redundantly in PTI signaling. We observed that flg22 induced phosphorylation of BES1 in the bik1/pbl1 double mutant (Fig. 3A). We previously reported that BIK1K105E, an ATP binding site mutant of BIK1, has a dominant-negative effect on PTI signaling (Zhang et al., 2010). Hence, we further examined whether BES1 phosphorylation induced by flg22 could be prevented by BIK1K105E. Consistent with the result of bik1/pbl1 double mutant, overexpression of BIK1K105E did not prevent flg22-induced BES1 phosphorylation (Fig. 3B), suggesting that BIK1 and PBL1 are not required for BES1 phosphorylation induced by flg22.

Figure 3.

Figure 3.

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BES1 phosphorylation induced by flg22 occurs downstream of MAPK activation. A, BIK1 and PBL1 are not required for BES1 phosphorylation induced by flg22. B, BIK1K105E does not prevent BES1 phosphorylation induced by flg22. C, BES1 phosphorylation induced by flg22 is prevented by HopAI1. D, BES1 phosphorylation induced by flg22 is partially blocked by MKK5K99M. Protoplasts isolated from wild-type (WT) or mutant plants were transfected with 35S::BES1-FLAG or together with 35S::BIK1K105E-hemagglutinin (HA), 35S::HopAI1-FLAG, or 35S::MKK5K99M-HA as indicated, treated with or without 1 μm flg22 for 5 min. Protein samples were separated by SDS-PAGE and subjected to anti-FLAG immunoblot. Coomassie Brilliant Blue (CBB) staining of the filter indicates loading of the protein.

Next, we checked MAPK activation induced by flg22 in bes1 mutants. Extent of MAPK activation induced by flg22 was comparable in bes1-1, bes1-2, and wild-type plants (Supplemental Fig. S3), suggesting that flg22-induced BES1 phosphorylation is likely to be either downstream or independent of MAPK activation. This promoted us to test whether MAPK activation is required for BES1 phosphorylation induced by flg22. In a previous study, we identified the unique phospho-Thr lyase activity of a P. syringae effector HopAI1 toward MPKs (Zhang et al., 2007, 2012), leading to permanent inactivation of MPK3, MPK6, and MPK4. Thus, HopAI1 was used as a bacterial inhibitor to block MAPK activation by flg22 in vivo. We observed that BES1 phosphorylation induced by flg22 was abolished in the presence of HopAI1 (Fig. 3C). MKK5K99M acts as a dominant negative mutant that partially blocks MAPK activation and downstream PTI signaling (Asai et al., 2002). Coexpression of MKK5K99M with BES1 also inhibited BES1 phosphorylation induced by flg22 (Fig. 3D). These results suggested that flg22-induced BES1 phosphorylation occurs downstream of MAPK activation.

MEKK1-MKK1/MKK2-MPK4 and MKK4/MKK5-MPK3/MPK6 constitute two MAPK cascade branches in PTI signaling. To determine whether MEKK1-MKK1/MKK2-MPK4 branch is responsible for BES1 phosphorylation, the effect of MPK4 or MKK1/MKK2 mutation on flg22-induced BES1 phosphorylation was examined. mpk4 and mkk1/mkk2 mutant displayed dwarf and autoimmune phenotype (Petersen et al., 2000). Mutation of SUPPRESSOR OF MKK1 MKK2 (SUMM2), a resistance gene that guards the MEKK1-MKK1/MKK2-MPK4 cascade, is able to suppress the autoimmune phenotype of mpk4 and mkk1/mkk2 mutant (Zhang et al., 2012). Thus, BES1 phosphorylation induced by flg22 was examined in mpk4/summ2 and mkk1/mkk2/summ2 mutants. As shown in Supplemental Figure S4, A and B, flg22 could induce BES1 phosphorylation in both mpk4/summ2 double mutant and mkk1/mkk2/summ2 triple mutant, indicating that mutation of MPK4 or MKK1/MKK2 is not sufficient to prevent BES1 phosphorylation induced by flg22. Expression of MKK5DD, a constitutive active form of MKK5, has been shown to activate MPK3/MPK6 in Arabidopsis protoplasts (Asai et al., 2002). BES-FLAG was then coexpressed with MKK5DD in Arabidopsis protoplasts, and a slower mobility shift of BES1 was detected in the presence of MKK5DD (Fig. 4A), indicating that activation of MPK3/MPK6 by MKK5DD is sufficient to induce BES1 phosphorylation in the absence of PAMP perception. Previous study also showed that MKK5K99M inhibited BES1 phosphorylation induced by flg22 (Fig. 3D); hence, we concluded that BES1 phosphorylation triggered by PAMP occurs downstream of MKK4/MKK5-MPK3/MPK6 branch. Subsequently, we examined BES1 phosphorylation induced by flg22 in mpk3 and mpk6 single mutants. A slight reduction of BES1 phosphorylation induced by flg22 in mpk6 mutant was detected by both regular SDS-PAGE (Fig. 4B) and Phos-tag SDS-PAGE followed by immunoblot (Fig. 4C). In the signaling pathway regulating stomatal patterning, MPK6AEF is a dominant negative mutant form that overcomes the redundancy of MPK3. We then further checked the effect of MPK6AEF on PAMP-triggered BES1 phosphorylation. As shown in Figure 4D, expression of MPK6AEF, but not of MPK6, inhibited BES1 phosphorylation induced by flg22. The above results indicated that BES1 phosphorylation induced by flg22 is likely to occur downstream of MPK3/MPK6 branch. The partial reduction of BES1 phosphorylation induced by flg22 in mpk6 mutant could be attributed to a putative functional redundancy with MPK3 or other MPKs. Normal BES1 phosphorylation induced by flg22 in mpk3 mutant indicated that MPK3 is not essential for BES1 phosphorylation triggered by PAMP (Supplemental Fig. S4C).

Figure 4.

Figure 4.

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MPK6 activity is required for BES1 full phosphorylation induced by flg22. A, Coexpression of MKK5DD enhances BES1 phosphorylation. B and C, BES1 phosphorylation induced by flg22 is partially affected in mpk6 mutant. D, Expression of MPK6AEF inhibits flg22-induced BES1 phosphorylation. Protoplasts isolated from wild-type (WT) or mutant plants were transfected with 35S::BES1-FLAG or together with 35S::MKK5DD-HA, 35S::MPK6-HA, or 35S::MPK6AEF-HA as indicated, treated with or without 1 μm flg22 for the indicated time. Protein samples were separated by SDS-PAGE (B) or Phos-tag SDS-PAGE (C) gel and subjected to anti-FLAG, anti-MPK6, or anti-phosphorylated extracellular signal-regulated kinase (pERK) immunoblot. Coomassie Brilliant Blue (CBB) staining of the filter indicates loading of the protein.

BES1 Is a Direct Substrate of MPK6

The above results raised the possibility of a physical protein-protein interaction between BES1 and MPK6. Purified glutathione S-transferase (GST), GST-tagged MPK6, MPK3, or MPK4 were incubated with an equal amount of purified His-BES1 and subjected to in vitro pull-down assay. GST-MPK6, but not GST-MPK3/MPK4 or GST, was detected to copurify with His-BES1 (Fig. 5A), indicating a direct and specific interaction between BES1 and MPK6 in vitro. The quantitative luciferase (LUC) complementation imaging assay was carried out to verify the specific interaction between BES1 and MPK6 in Nicotiana benthamiana. Coexpression of N-terminal of luciferase (NLuc)-tagged BIK1 and C-terminal of luciferase (CLuc)-tagged AtrbohD driven by 35S promoter was used as control for a positive interaction (Li et al., 2014). Coexpression of NLuc-MPK6 and CLuc-BES1 driven by 35S promoter in N. benthamiana resulted in much higher luciferase activity compared with coexpression of NLuc-MPK6 and CLuc-BIK1, NLuc-MPK6 and CLuc vector, or CLuc-BIK1 and NLuc vector (Fig. 5, B and C), indicating a specific interaction between BES1 and MPK6 in vivo. Expression levels of NLuc- and CLuc-fusion proteins were detected by immune blot (Supplemental Fig. S5). Thus, it was shown that MPK6 is able to interact with BES1 both in vitro and in vivo.

Figure 5.

Figure 5.

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MPK6 interacts with and phosphorylates BES1. A, GST-MPK6 interacts with His-BES1 in vitro. An equal amount of His-BES1 was incubated with GST, GST-MPK6, GST-MPK3, or GST-MPK4, precipitated with glutathione agarose, and western-blot (WB) analysis was used to detect the presence of His-BES1. The amount of GST or His-tagged protein was determined by anti-GST or anti-His immunoblot. The experiment was repeated three times with similar results. B, Luciferase imaging of MPK6 and BES1 interaction in N. benthamiana. N. benthamiana leaves infiltrated with 35S::NLuc-MPK6, 35S::CLuc-BES1, 35S::CLuc-BIK1, 35S::CLuc-AtrbohD, 35S::NLuc-BIK1, 35S::NLuc, or 35S::CLuc empty vector (EV) as indicated were subjected to luciferase complementation imaging assay. C, Quantitative luminescence of MPK6 and BES1 interaction in N. benthamiana. N. benthamiana leaves infiltrated with indicated constructs were sliced into strips, and relative luminescence was determined by a microplate luminometer. Error bars indicate sd of three technical repeats. Asterisks indicate significant difference at P < 0.01. Similar results were observed in three independent biological repeats. NLuc, N-terminal fragment of firefly luciferase; CLuc, C-terminal fragment of firefly luciferase. D, BES1 is phosphorylated by MPK6 in vitro. GST-BES1, GST-MPK6, or GST-MPK6Km alone and GST-BES1 incubated with GST-MPK6 or GST-MPK6Km are subjected to in vitro phosphorylation assay. Coomassie Brilliant Blue (CBB) staining indicates loading of the protein. GST-MPK6Km stands for MPK6 K92R mutation.

We then speculated a direct phosphorylation of BES1 by MPK6. In vitro phosphorylation assay was conducted to test whether BES1 was a direct substrate of MPK6. Purified His-BES1 or GST-BES1 alone did not exhibit autophosphorylation activity, whereas addition of purified GST-MPK6 resulted in phosphorylation of both His-BES1 and GST-BES1 in vitro (Supplemental Fig. S6). Meanwhile, GST-MPK6Km, a kinase inactive mutant of MPK6, failed to phosphorylate BES1 in vitro (Fig. 5D). Thus, we concluded that BES1 is a direct substrate of MPK6 in vitro.

It is known that phosphorylation of BES1 by GSK3-like kinase BIN2 induce its nuclear export in BR signaling (Yin et al., 2002; Ryu et al., 2010). We next studied the effect of MAPK-mediated BES1 phosphorylation on its subcellular accumulation (Supplemental Materials and Methods S2). Expression of MKK5DD, which phosphorylates MPK6 to constitutively activate PTI (Asai et al., 2002) and enhances BES1 phosphorylation, reduced the relative nucleus to cytoplasm ratio of BES1-GFP (Supplemental Fig. S7, A and B). The results suggested that MAPK-mediated phosphorylation alters BES1 subcellular accumulation in PTI.

BES1 S286 and S137 Residues Are Required for Full Phosphorylation Induced by flg22 and Bacterial Resistance

To determine BES1 phosphorylation sites targeted by activated MPK6, His-BES1 was incubated with MPK6 activated by MKK5DD (Asai et al., 2002). Phosphorylated BES1 was fractionated by SDS-PAGE and subjected to mass spectrometry analysis (Supplemental Materials and Methods S3). Several putative phosphorylation sites targeted by MPK6 were identified in BES1 (Supplemental Fig. S8). Because MAPKs are Pro-directed kinases (Kyriakis and Avruch, 2001), Ser residues that are immediately followed by Pro residue were selected as putative phosphorylation sites. Site-directed mutagenesis followed by mobility shift assay was conducted to identify critical sites required for BES1 phosphorylation induced by flg22. Of the potential residues identified by mass spectrometric analysis, BES1S286A mutation significantly impaired flg22-induced BES1 phosphorylation when BES1S286A-FLAG was transiently expressed in protoplasts (Fig. 6A). Double mutation of BES1 S286A and S137A residues (BES1SSAA) almost completely blocked BES1 phosphorylation induced by flg22 (Fig. 6A). We next studied in vivo phosphorylation of BES1 wild type and BES1SSAA protein in BES1::BES1-FLAG and BES1::BES1SSAA-FLAG transgenic plants, respectively. In contrast to normal BES1 phosphorylation induced by flg22 in BES1::BES1-FLAG transgenic plants pretreated with or without epiBL, a significantly compromised BES1SSAA phosphorylation induced by flg22 was detected in BES1::BES1SSAA-FLAG transgenic plants (Fig. 6B). The results indicated that S286 and S137 residues are required for flg22-induced BES1 full phosphorylation in vivo, in which S286 plays a greater role than S137.

Figure 6.

Figure 6.

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BES1 S286 and S137 are required for flg22-induced full phosphorylation. A, S286 and S137 are required for BES1 full phosphorylation induced by flg22 in protoplasts. Protoplasts were transfected with 35S::BES1-FLAG, 35S::BES1S286A-FLAG, 35S::BES1S137A-FLAG, or 35S::BES1SSAA-FLAG as indicated, treated with or without flg22. B, S286 and S137 are required for BES1 full phosphorylation induced by flg22 in transgenic plants. BES1::BES1-FLAG or BES1::BES1SSAA-FLAG transgenic plants were pretreated with or without 1 μm epiBL as indicated for 1 h, followed by 1 μm flg22 treatment. Protein samples were subjected to anti-FLAG immunoblot. Coomassie Brilliant Blue (CBB) or Ponceau S (PS) staining of the filter indicates loading of the protein. WT, Wild type.

BES1SSAA Impairs Plant Bacterial Resistance But Not BR-Mediated Hypocotyl and Root Growth

To determine the contribution of PAMP-induced BES1 phosphorylation in plant resistance, bes1-1/BES1::BES1-FLAG and bes1-1/BES1::BES1SSAA-FLAG plants were constructed (Supplemental Fig. S9) and in planta bacterial growth of Pst DC3000 was examined in these transgenic lines (Fig. 7A). Hypocotyl length of bes1-1 mutants was shorter than that of wild-type plants in response to brassinolide (BL) treatment (He et al., 2005), while hypocotyl length was comparable in bes1-1/BES1::BES1-FLAG plants and wild-type plants in response to BL (Fig. 7B), indicating that BES1::BES1-FLAG protein was functional. BES1::BES1-FLAG, but not BES1::BES1SSAA-FLAG, could restore bacterial resistance of bes1-1 mutant against Pst DC3000 (Fig. 7A), indicating that BES1 S286 and S137 are critical residues required both for phosphorylation triggered by PAMP and for full resistance against bacterial pathogen. Expression of BES1::BES1-FLAG and BES1::BES1SSAA-FLAG in transgenic lines was confirmed by immune blot (Supplemental Fig. S10). As BR triggers dephosphorylation of BES1, an altered BR response in bes1-1 mutant complemented with BES1SSAA would be expected if BES1 S286 and S137 residues are also required for BR signaling. To check the effect of BES1SSAA mutation on BR-mediated plant growth, hypocotyl and root length of bes1-1 mutant complemented with BES1 wild type or BES1SSAA both in the absence or presence of epiBL was further examined. In contrast to a compromised bacterial resistance in bes1-1/BES1::BES1SSAA-FLAG plants, a normal hypocotyl and root length was detected in bes1-1/BES1::BES1SSAA-FLAG plants compared with bes1-1/BES1::BES1-FLAG plants, both in the absence or presence of epiBL (Fig. 7, B and C). We further examined the expression of BR-responsive genes in bes1-1 mutant, bes1-1/BES1::BES1-FLAG, and bes1-1/BES1::BES1SSAA-FLAG plants (Supplemental Materials and Methods S4). A compromised induction of PACLOBUTRAZOL RESISTANCE1 (PRE1) and PRE5 by epiBL was observed in bes1-1 mutant (Supplemental Fig. S11, A and B). Both BES1-FLAG and BES1SSAA-FLAG could restore PRE1 and PRE5 induction by epiBL in bes1-1 mutant (Supplemental Fig. S11). The results indicated that BES1 S286 and S137 residues are not essential for BR-mediated plant growth. This is in agreement with previous reports that BES1 S129, S133, S171, and T175 are critical residues required for BR signaling (Ryu et al., 2007, 2010). Although the molecular mechanism by which BES1 phosphorylation regulates downstream PTI responses remains to be further clarified, different residue requirements of BES1 in PTI and BR signaling suggest differential modulation of a common transcription factor by plant hormone and immune signals. Additionally, the assumption that HDMs are recruited by BES1 to control defense-related gene expression is of interest for further investigation.

Figure 7.

Figure 7.

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BES1SSAA impairs bacterial resistance to Pst DC3000 but not BR-mediated hypocotyl and root growth. A, S286 and S137 are required for full resistance to Pst DC3000. Growth of Pst DC3000 in wild-type (WT), bes1-1, bes1-1 complemented with BES1, or BES1SSAA lines. The results shown are representative of three independent biological repeats. Error bars indicate sd of four technical repeats. Asterisk indicates significant difference to wild-type plants at P < 0.05. B and C, S286 and S137 are not required for BR-mediated hypocotyl (B) and root (C) growth. Hypocotyl and root lengths of long-day-grown wild-type, bes1-1, bes1-1 complemented with BES1, or BES1SSAA seedlings on medium containing 0 or 100 nm epiBL. Data are means ± se (n ≥ 12). Asterisks indicate significant difference to the 100 nm BL-treated wild type at P < 0.01. Similar results were observed in three independent biological repeats. GFP fluorescent BES1::BES1-FLAG and BES1::BES1SSAA-FLAG T3 transgenic seeds were used for bacterial growth assay and hypocotyl and root growth assay.

DISCUSSION

PAMP recognition by PRR complex activates signaling kinases, such as MAPKs, calcium-dependent protein kinases, and BIK1/PBLs, to regulate downstream defense responses. However, only a few direct substrates of MAPKs in plant immune signaling have been identified, such as MKS1, AtPHOS32, AtERF104, VIP1, ACS2, and ACS6 (Peck et al., 2001; Liu and Zhang, 2004; Andreasson et al., 2005; Djamei et al., 2007; Merkouropoulos et al., 2008; Bethke et al., 2009; Han et al., 2010; Li et al., 2012). In addition, AtERF5 and AtERF6 transcription factors have also been reported as direct substrates of MPK6 and/or MPK3 that regulate plant immunity against fungal and bacterial pathogens, whereas their function in PTI is uncertain (Moffat et al., 2012; Son et al., 2012; Meng et al., 2013). Proteomic and phosphoproteomic approaches have been used to identify putative substrates for MAPKs in vivo; however, their biological significance requires further investigation (Peck et al., 2001; Merkouropoulos et al., 2008; Hoehenwarter et al., 2013). In this study, we show that BES1 is a direct substrate of MPK6 in PTI. In contrast to a reduced phosphorylation of BES1 in response to the plant hormone BR (Wang et al., 2002; Yin et al., 2005), an elevated BES1 phosphorylation upon bacterial PAMP perception was observed. PAMP perception triggers both the activation of MAPK cascades and the phosphorylation of BIK1/PBL1. We have previously shown that BIK1 and PBL1 are not required for MAPK activation in response to flg22 treatment (Feng et al., 2012), suggesting that BIK1/PBL1 phosphorylation and MAPK activation act in two branches. We found that PAMP-triggered BES1 phosphorylation is independent of BIK1 and PBL1, but it is suppressed when MAPK activity is inhibited. Hence, the results suggested a MAPK-dependent BES1 phosphorylation triggered by PAMP perception. Protein-protein interaction and protein phosphorylation studies demonstrate that BES1 associates with MPK6 and is phosphorylated by MPK6 upon PAMP perception. In addition, a comparison of BES1-direct target genes (Yu et al., 2011) with flg22-regulated genes showed that 234 out of 3,286 (7.1%) flg22-regulated genes (Chen et al., 2009) are BES1-direct target genes (14.5% of 1,609; Supplemental Fig. 12), indicating an important role of BES1 in PTI signaling.

We observed an elevated BES1 phosphorylation induced by flg22 both in Arabidopsis protoplasts and in 5-week-old mature transgenic plants grown under short-day conditions and treated with flg22 for 5 min. In a previous study, flg22 had been shown not to affect BES1 phosphorylation in 2-week-old transgenic seedlings treated with flg22 for 1.5 h (Albrecht et al., 2012). In addition to different plant growth conditions, the phosphorylation status of BES1 is also regulated by BR levels in plants and is affected by various environmental signals. Given that BR levels vary in plants throughout developmental stages, the phosphorylation status of BES1 may also vary. The conflicting results and different protein expression patterns in plants and protoplasts could be attributed to the differential BR levels due to developmental and environmental signals. Both in Arabidopsis protoplasts and in transgenic plants, Flg22 induced BES1 phosphorylation but not BES1SSAA phosphorylation (Fig. 6, A and B). The flg22-induced BES1 phosphorylation could be partially masked by basal level of phosphorylated BES1, which was accumulated in untreated plants. Even though BES1 was almost completely dephosphorylated by epiBL pretreatment, the following flg22 treatment clearly resumed BES1 phosphorylation in BES1::BES1-FLAG plants (Fig. 2B). These results demonstrate that PAMP induces BES1 phosphorylation at S286 and S137 residues in vivo and that the absence of flg22 effect on BES1SSAA phosphorylation is not a consequence of BR signaling preactivation.

Synergistic and antagonistic actions in plants constitute complex signal transduction networks between host immunity and hormone signaling that play an important role in balancing development with resistance. Recent studies revealed a complicated interplay between FLS2-mediated PTI signaling and BRI-mediated BR signaling pathways, which share a common coreceptor, BAK1 (Li et al., 2002; Wang and Chory, 2006; Chinchilla et al., 2007; Heese et al., 2007; Wang et al., 2008; Fan et al., 2014; Malinovsky et al., 2014). Both excess and reduced BR biosynthesis significantly compromised flg22-induced responses (Belkhadir et al., 2012). In addition to antagonistic and synergistic effect conferred by BAK1 competition, BR was also shown to inhibit PTI signaling either downstream or independently of BAK1 and BIK1 (Albrecht et al., 2012; Belkhadir et al., 2012). Additionally, BR-signaling kinase1 was reported to interact with FLS2 and regulate a subset of PTI signaling (Tang et al., 2008; Shi et al., 2013), while BIK1 was shown to negatively regulate BR signaling (Lin et al., 2013). BRASSINAZOLE RESISTANT1 cooperates with WRKY transcription factors and negatively regulates early PTI responses (Lozano-Durán et al., 2013). In this study, we demonstrate that MAPK-mediated BES1 phosphorylation induced by PAMP perception regulates plant immunity positively, which uncovers a different modulation of BES1 from the GSK3-like kinase BIN2-mediated BES1 phosphorylation in BR signaling (Yin et al., 2002, 2005; Ryu et al., 2010).

Perception of bacterial flagellin led to an elevation of BES1 phosphorylation. However, MAPK inactivation, via the expression of HopAI1, MKK5K99M, or MPK6AEF, inhibited PAMP-induced BES1 phosphorylation. The results indicate that PAMP perception enhances the phosphorylation of BES1 through pathogen-induced MAPKs. bes1 mutant was compromised in resistance to bacterial pathogen and showed shorter hypocotyl length than wild-type plants in response to BL treatment (He et al., 2005). BES1SSAA restored BR-mediated plant growth but failed to restore the compromised resistance in bes1 mutant (Fig. 7; Supplemental Figure S11), indicating that BES1 phosphorylation positively regulates plant immunity and that BES1 S286 and S137 phosphorylation was required for PTI but not for BR signaling. On the other hand, BES1 S129, S133, S171, and T175 have been identified as critical residues required for BR signaling (Ryu et al., 2007, 2010). These results indicate that differential modulation of BES1 phosphorylation is achieved by distinct upstream kinases in PTI and BR signaling. BES1 has also been demonstrated to be a substrate of more axillary growth locus2 (MAX2), a subunit of the S phase kinase-associated protein1-cullin-F box type ubiquitin E3 ligase, which regulates SL-responsive gene expression (Wang et al., 2013). The degradation rate of phosphorylated and dephosphorylated BES1 by MAX2 is different (Wang et al., 2013), suggesting a putative effect of BES1 phosphorylation status on SL signaling. Whether BES1SSAA mutation affects shoot branching is of interest for further investigation. Overall, these results indicate diverse modulation of BES1 targeted by distinct signaling proteins and a broad role of a common transcription factor in multiple hormones and immunity signaling pathways.

MATERIALS AND METHODS

Plants, Constructs, and Antibodies

Arabidopsis (Arabidopsis thaliana) plants used in this study include the Col-0 wild type and the bes1-1 (He et al., 2005), bes1-2 (Supplemental Fig. S1A), bik1/pbl1 (Zhang et al., 2010), summ2, mpk4/summ2, mkk1/mkk2/summ2 (Zhang et al., 2012), mpk3, and mpk6-2 (Zhang et al., 2007) mutants. Constructs used in this study include MKK5DD-His (Zhang et al., 2007), 35S-BIK1K105E-HA (Zhang et al., 2010), and 35S-AtrbohD-Cluc (Li et al., 2014). pUC-FLAG (Li et al., 2005), pUC-Nluc, pUC-Cluc, pCambia1300-Nluc, and pCambia1300-Cluc (Chen et al., 2008) plasmid vectors were used to generate transient expression or transgenic expression constructs. The pFAST-G01 vector (Shimada et al., 2010) harboring a fluorescent OLEOSIN1-GFP marker that is specifically expressed on seed oil body membrane was used for BES1::BES1-FLAG and BES1::BES1SSAA-FLAG transgenic plant construction, providing an immediate and nondestructive way of identifying transformed seeds under fluorescence microscopy. Other plants, constructs, and antibodies used were described in supporting information.

Bacterial Growth Assay

Five-week-old plants grown under short-day conditions were infiltrated with 105 cfu mL–1 Pst DC3000. Leaf bacterial number was determined at indicated days post inoculation. Each data point consisted of at least three replicates. GFP fluorescent BES1::BES1-FLAG and BES1::BES1SSAA-FLAG T3 transgenic seeds were used for bacterial growth assay, and the expression of BES1::BES1-FLAG and BES1::BES1SSAA-FLAG protein was further confirmed by immune blot.

Quantitative Reverse Transcription-PCR

Five-week-old plants were treated with or without 500 nm flg22 or 100 nm epiBL as indicated for 3 h. Total RNA was extracted with TRIZOL (Invitrogen) and used for reverse transcription. Real-Time PCR was performed by using SYBR Premix Ex Taq kit (TaKaRa) following standard protocols. The housekeeping gene TUBLIN was used as internal control.

Transient Expression in Protoplast

Protoplasts isolated from 5-week-old plants were transfected with the indicated constructs. Twelve hours after transfection, protoplasts were treated with 1 μm flg22 or water as indicated for 5 min.

Detection of BES1 Phosphorylation in Protoplast and Plant

Protoplasts transfected with constructs as indicated were treated with 1 μm flg22 or water. Five-week-old BES1::BES1-FLAG transgenic plants were infiltrated with 1 μm flg22 or water. One micromolar epiBL was infiltrated into plants 1 h before flg22 treatment in BL-pretreated experiments. For phosphatase treatment, total protein was treated with λ PPase (New England Biolabs) according to manufacturer’s instruction. In Phos-tag SDS-PAGE assay, SDS-PAGE gel supplied with 20 μm Phos-tag was used for electrophoresis. Total protein was extracted and subjected to anti-FLAG immunoblot.

GST Pull-Down Assay

GST, GST-MPK6, GST-MPK3, GST-MPK4, or His-BES1 was expressed in Escherichia coli and purified. An equal amount of the purified His-BES1 was incubated with GST, GST-MPK6, GST-MPK3, or GST-MPK4 as indicated for 4 h and then passed through the glutathione agarose column. The column was washed four times, and bound protein was boiled and separated by SDS-PAGE gel.

Luciferase Complementation Imaging Assay

Agrobacterium tumefaciens strains carrying CLuc and NLuc constructs were mixed and infiltrated into leaves of Nicotiana benthamiana. Leaves coexpressing different constructs were examined for LUC activity 2 d after infiltration. N. benthamiana leaves were kept in dark for 5 min after adding 1 mm luciferin to quench the fluorescence. LUC image was captured by cooled CCD imaging apparatus (Roper Scientific). Quantitative LUC activity was determined by Microplate Luminometer (Promega). Expression of CLuc-tagged proteins and NLuc-tagged proteins was detected by anti-CLuc or anti-Luc immunoblot.

Hypocotyl and Root Growth Assay

Arabidopsis Col-0 wild-type, bes1-1 mutant, and GFP fluorescent BES1::BES1-FLAG and BES1::BES1SSAA-FLAG T3 transgenic seeds were surface sterilized and plated on one-half-strength Murashige and Skoog medium supplemented with 9 g L–1 agar and 10 g L–1 Suc with or without epiBL as indicated. Seedlings were grown under long-day conditions for 6 d after 2 d of incubation at 4°C, and hypocotyl and root length was measured.

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_167_3_1076__index.html (1.2KB, html)

Acknowledgments

We thank Jian-min Zhou (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for sharing biological materials and providing reagents, Yuelin Zhang (University of British Columbia) for summ2, mpk4/summ2, and mkk1/mkk2/summ2 seeds, and Daoxin Xie (Tsinghua University) for bes1-1 seeds.

Glossary

PAMP

pathogen-associated molecular pattern

PRR

pattern recognition receptor

PTI

pathogen-associated molecular pattern-triggered immunity

MAPK

mitogen-activated protein kinase

BR

brassinosteroid

HDM

histone demethylase

SL

strigolactone

Pst DC3000

Pseudomonas syringae pv tomato DC3000

Col-0

ecotype Columbia

PPase

protein phosphatase

GST

glutathione S-transferase

Footnotes

1

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB11020600) and the Chinese Natural Science Foundation (grant no. 31300234).

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