The Arabidopsis transcription factor BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 is a direct substrate of MITOGEN-ACTIVATED PROTEIN KINASE6 and regulates 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 (BES1(SSAA)) 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, BES1(SSAA) 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.

Figure 1.

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.

Figure 2.

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.

Figure 3.

BES1 phosphorylation induced by flg22 occurs downstream of MAPK activation. A, BIK1 and PBL1 are not required for BES1 phosphorylation induced by flg22. B, BIK1^K105E 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 MKK5^K99M. Protoplasts isolated from wild-type (WT) or mutant plants were transfected with 35S::BES1-FLAG or together with 35S::BIK1^K105E-hemagglutinin (HA), 35S::HopAI1-FLAG, or 35S::MKK5^K99M-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.

Figure 4.

MPK6 activity is required for BES1 full phosphorylation induced by flg22. A, Coexpression of MKK5^DD enhances BES1 phosphorylation. B and C, BES1 phosphorylation induced by flg22 is partially affected in mpk6 mutant. D, Expression of MPK6^AEF inhibits flg22-induced BES1 phosphorylation. Protoplasts isolated from wild-type (WT) or mutant plants were transfected with 35S::BES1-FLAG or together with 35S::MKK5^DD-HA, 35S::MPK6-HA, or 35S::MPK6^AEF-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.

Figure 5.

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-MPK6^Km alone and GST-BES1 incubated with GST-MPK6 or GST-MPK6^Km are subjected to in vitro phosphorylation assay. Coomassie Brilliant Blue (CBB) staining indicates loading of the protein. GST-MPK6^Km stands for MPK6 K92R mutation.

Figure 6.

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::BES1^S286A-FLAG, 35S::BES1^S137A-FLAG, or 35S::BES1^SSAA-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::BES1^SSAA-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.

Figure 7.

BES1^SSAA 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 BES1^SSAA 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 BES1^SSAA 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::BES1^SSAA-FLAG T3 transgenic seeds were used for bacterial growth assay and hypocotyl and root growth assay.