BR-SIGNALING KINASE1 physically associates with FLAGELLIN SENSING2 and regulates plant innate immunity in Arabidopsis

Abstract

Pathogen-associated molecular pattern (PAMP)-trigged immunity (PTI) is the first defensive line of plant innate immunity and is mediated by pattern recognition receptors. Here, we show that a mutation in BR-SIGNALING KINASE1 (BSK1), a substrate of the brassinosteroid (BR) receptor BRASSINOSTEROID INSENSITIVE1, suppressed the powdery mildew resistance caused by a mutation in ENHANCED DISEASE RESISTANCE2, which negatively regulates powdery mildew resistance and programmed cell death, in Arabidopsis thaliana. A loss-of-function bsk1 mutant displayed enhanced susceptibility to virulent and avirulent pathogens, including Golovinomyces cichoracearum, Pseudomonas syringae, and Hyaloperonospora arabidopsidis. The bsk1 mutant also accumulated lower levels of salicylic acid upon infection with G. cichoracearum and P. syringae. BSK1 belongs to a receptor-like cytoplasmic kinase family and displays kinase activity in vitro; this kinase activity is required for its function. BSK1 physically associates with the PAMP receptor FLAGELLIN SENSING2 and is required for a subset of flg22-induced responses, including the reactive oxygen burst, but not for mitogen-activated protein kinase activation. Our data demonstrate that BSK1 is involved in positive regulation of PTI. Together with previous findings, our work indicates that BSK1 represents a key component directly involved in both BR signaling and plant immunity.

Figure 1.

Suppression of the edr2-Mediated Phenotypes by bsk1-1. (A) Four-week-old Arabidopsis plants were infected with G. cichoracearum. The plants were photographed at 8 DAI. The wild-type plants were susceptible, and a large number of spores were produced on the leaves. By contrast, the edr2 mutant was resistant, displaying massive necrotic cell death and very few spores. The edr2 bsk1-1 plants displayed a wild-type-like susceptible phenotype, and no visible necrotic lesions were present on the leaves. A genomic clone containing the BSK1 gene complemented the bsk1-1 mutation. WT, the wild type. (B) Fungal growth and cell death on 4-week-old plants at 8 DAI with G. cichoracearum. Leaves were stained with trypan blue to show fungal structures and dead cells. A large number of spores were produced in the wild type and edr2 bsk1-1, while extensive mesophyll cell death occurred in edr2. Bar = 50 μm (C) Quantification of fungal growth in plants at 5 DAI by counting the number of conidiophores per colony. Results were from one experiment, and the bars represent mean and sd (n > 30). Statistically significant differences are indicated by lowercase letters (P < 0.05; one-way analysis of variance [ANOVA]). The experiments were repeated three times with similar results. (D) Infected leaves were stained with 3,3′-diamino benzidine-HCl and trypan blue sequentially at 2 DAI to visualize H₂O₂ (brown staining) and fungal structures (blue staining). Bar = 20 μm (E) and (F) The bsk1-1 mutation suppressed enhanced ethylene-induced senescence in edr2. Four-week-old plants were exposed to 100 μL L⁻¹ ethylene for 3 d, and increased chlorosis was observed in edr2 mutants after ethylene treatment. The edr2 bsk1-1 plants showed a wild-type-like phenotype (E). The chlorophyll content in leaves from the ethylene treated plants (F). Bars represent sd of values obtained from five plants. Lowercase letters represent significant difference from the wild type (P < 0.01, one-way ANOVA). Three independent experiments were performed with similar results. FW, fresh weight. (G) The bsk1-1 mutation was identified by standard map-based cloning. Markers and BAC clones are indicated. (H) Structure of the BSK1 gene. The asterisk indicates the bsk1-1 mutation. Exons are indicated by black boxes and introns by black lines. A nucleotide change (G1328A) was identified in the BSK1 gene and leads to an amino acid substitution (R443Q) in the BSK1 protein. The BSK1 protein contains a kinase domain and a TPR domain. The bsk1-1 point mutation (asterisk) is in the TPR domain. aa, amino acids.

Figure 2.

Responses of the bsk1-1 Mutant to Pathogens. (A) to (C) Four-week-old plants were infected with Pto DC3000 (A), Pto DC3000 avrRpt2 (B), and Pto DC3000 avrPphB (C). The plants were inoculated with bacterial suspensions at OD₆₀₀ = 0.0005. The number of bacteria was counted at 4 h after infection and 3 DAI. cfu, colony-forming units; WT, the wild type. (D) Two-week-old plants were infected with H. a. Noco 2. The number of sporangiophores was counted at 7 DAI. Bars represent sd of values obtained from three independent samples. Lowercase letters represent significant differences from the wild type (P < 0.05, one-way ANOVA). At least three independent experiments were performed with similar results.

Figure 3.

SA Levels Are Affected by the bsk1-1 Mutation. Four-week-old plants were infected with G. cichoracearum or Pto DC3000. (A) Accumulation of PR1 transcripts was examined by quantitative real-time PCR at various time points after inoculation with Pto DC3000 (OD₆₀₀ = 0.001). ACTIN2 was used as an internal control. Bars represent mean and sd from three independent experiments. Statistically significant differences are indicated by one asterisk (P < 0.05, Student’s t test) or two asterisks (P < 0.01, Student’s t test). hpi, h after infection; WT, the wild type. (B) Free SA levels were measured in the uninfected and infected (3 DAI) leaves after inoculation with G. cichoracearum. FW, fresh weight. (C) Free SA levels were measured in the 10 mM MgCl₂ treated or Pto DC3000 (OD₆₀₀ = 0.001) infected (2 DAI) leaves. (B) and (C) Bars represent mean and sd from three independent biological experiments. Lowercase letters indicate statistically significant differences (P < 0.01, one-way ANOVA).

Figure 4.

BSK1-GFP Localizes to the Plasma Membrane, and Disruption of the Predicted Myristoylation Site Compromises BSK1 Function. (A) BSK1-GFP was transformed into Arabidopsis protoplasts, and the GFP signal was detected by confocal microscopy. BSK1-GFP localizes to cell periphery, and disruption of the myristoylation site of BSK1 (G2A) compromised BSK1 cell periphery localization. EV, empty vector. (B) Subcellular fractionation and immunoblot assays. Total protein was extracted from 4-week-old plants. Total (T), soluble (S), and membrane (M) fractions of protein from BSK1-HA or BSK1 G2A-HA transgenic plants were loaded on SDS-PAGE gels and subjected to immunoblotting with anti-HA antibody. GAPDH and H⁺-ATPase were used as soluble marker or plasma membrane marker, respectively. Molecular masses of protein markers are shown on the right. Ponceau S staining of ribulose-1,5-bis-phosphate carboxylase/oxygenase is shown as a loading control. The experiments were repeated three times with similar results. WT, the wild type. (C) to (E) The BSK1 G2A-HA clone was unable to restore edr2 bsk1-1 to the edr2 phenotype. Four-week-old plants were infected with G. cichoracearum. (C) Representative leaves were removed and photographed at 8 DAI. Leaves of two independent transgenic lines for each construct are shown. More than 10 independent transgenic lines expressing the fusion proteins of the correct sizes were examined for each construct. All the transgenic lines examined showed consistent phenotypes. (D) The infected leaves at 8 DAI were stained with trypan blue. Bar = 100 μm. (E) The number of conidiophores per colony was counted at 5 DAI. Bars represent mean and sd (n > 30). Lowercase letters represent statistically significant differences (P < 0.01, one-way ANOVA). The experiments were repeated three times with similar results.

Figure 5.

BSK1 Has Kinase Activity in Vitro, and the Kinase Activity Is Required for Defense Function. (A) BSK1 can autophosphorylate in vitro. Recombinant maltose binding–BSK1 fusion protein (MBP-BSK1) or MBP-BSK1 (K104E) was incubated in a kinase assay buffer containing [γ-³²P]ATP and 10 mM MnCl₂ or 10 mM MgCl₂ as indicated, then separated by SDS-PAGE and detected by autoradiography. Kinase activity was assessed with a dose–response assay. First lane, 1 μg MBP; second lane, 2 μg MBP-BSK1; the next six lanes, 20 ng, 200 ng, or 2 μg of MBP-BSK1 or MBP-BSK1(K104E). Increasing amounts of protein are denoted with a triangle. Top panel, autoradiogram; bottom panel, Coomassie blue (CBB) staining. The molecular weight markers are indicated. The experiment was repeated three times with similar results. (B) BSK1 (K104E), the kinase-dead mutant form of BSK1, cannot complement the bsk1-1 mutation. The BSK1 or BSK1(K104E) genomic clone was transformed into edr2 bsk1-1 mutants, and the transgenic plants were infected with G. cichoracearum. Representative leaves were removed and photographed at 8 DAI (top panel). The infected leaves at 8 DAI were stained with trypan blue to visualize the fungal structures and mesophyll cell death in the leaves. WT, the wild type. Bar = 100 μm. (C) Quantification of fungal growth by counting the number of conidiophores per colony at 5 DAI. Bars represent mean and sd (n > 30). Lowercase letters indicate statistically significant differences (P < 0.01, one-way ANOVA). Experiments were repeated three times with similar results.

Figure 6.

BSK1 Forms a Protein Complex with FLS2. (A) Co-IP of BSK1 and FLS2 from N. benthamiana transiently expressing BSK1-FLAG and FLS2-YFP-HA before (−) or 10 min after (+) elicitation with 1 μM flg22, as indicated. Total protein was extracted and subjected to immunoprecipitation of BSK1 protein by FLAG antibody, followed by immunoblot analysis with anti-GFP antibody. BAK1-FLAG was used an internal control. Asterisk indicates the heavy chain of IgG (∼55 kD). (B) Co-IP of BSK1 and FLS2 from Arabidopsis protoplasts transiently expressing BSK1-GFP and FLS2-FLAG before (−) or 10 min after (+) elicitation with 1 μM flg22 as indicated. FLS2-FLAG alone was used as a negative control. The BSK1 protein was immunoprecipitated by GFP antibody, followed by immunoblot analysis with anti-FLAG antibody. BAK1-GFP was used as an internal control. (C) Co-IP of BSK1 and FLS2 from transgenic Arabidopsis plants. Total protein was extracted from 3-week-old plants expressing both BSK1-Myc and FLS2-YFP-HA. Plants expressing FLS2-YFP-HA alone (left panel) or an immunoprecipitation with unrelated antibody (right panel) were used as negative controls. The BSK1 protein was immunoprecipitated by anti-Myc antibody, and the presence of FLS2-YFP-HA protein was detected by immunoblot analysis with anti-HA antibody. (D) and (E) The bsk1-1 mutation did not affect BSK1 and FLS2 association in N. benthamiana or Arabidopsis protoplasts. Co-IP of BSK1 and FLS2 from N. benthamiana transiently expressing BSK1m-GFP (carrying the bsk1-1 mutation) and FLS2-YFP-HA (D) or Arabidopsis protoplasts transiently expressing BSK1m-GFP and FLS2-FLAG (E). BSK1-GFP or BSK1m-GFP alone was used as a negative control. Total protein was subjected to immunoprecipitation with anti-HA (D) or anti-FLAG (E) antibody, followed by immunoblot analysis using anti-GFP antibody. These experiments were repeated four times with similar results.

Figure 7.

bsk1-1 Displayed Defects in flg22-Induced ROS Burst. (A) Leaves of the wild type, bsk1-1, fls2, and two bsk1-1 complementation lines were treated with 100 nM flg22 and incubated with luminol and horseradish peroxidase to detect ROS. Luminescence was recorded at different time points as indicated. Error bars represent sd of data derived from replicate samples (n = 12). WT, the wild type. (B) Total photon counts during 30 min of treatment are presented to indicate the ROS production. Bars represent sd (n = 12). Statistically significant differences were indicated with lowercase letters (P < 0.01, one-way ANOVA). (C) Accumulation of BSK1 transcript in response to flg22. The wild-type seedlings were treated with 100 nM flg22 at different time points. hpi, h after infection. (D) The transcript accumulation of PR1 was examined by quantitative real-time PCR at various time points after treatment with 100 nM flg22. ACTIN2 was used as an internal control. Bars represent mean and sd from three independent experiments. Statistically significant difference is indicated by an asterisk (P < 0.05, Student’s t test). The experiments were repeated three times with similar results.