Brassinosteroids inhibit pathogen-associated molecular pattern-triggered immune signaling independent of the receptor kinase BAK1

Abstract

Plants and animals use innate immunity as a first defense against pathogens, a costly yet necessary tradeoff between growth and immunity. In Arabidopsis, the regulatory leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1 combines with the LRR-RLKs FLS2 and EFR in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and the LRR-RLK BRI1 in brassinosteroid (BR)-mediated growth. Therefore, a potential tradeoff between these pathways mediated by BAK1 is often postulated. Here, we show a unidirectional inhibition of FLS2-mediated immune signaling by BR perception. Unexpectedly, this effect occurred downstream or independently of complex formation with BAK1 and associated downstream phosphorylation. Thus, BAK1 is not rate-limiting in these pathways. BRs also inhibited signaling triggered by the BAK1-independent recognition of the fungal PAMP chitin. Our results suggest a general mechanism operative in plants in which BR-mediated growth directly antagonizes innate immune signaling.

Fig. 1.

EpiBL perception does not induce PTI responses. (A) Oxidative burst triggered by flg22, elf18, or epiBL in Col-0 leaf discs. ROS production is presented as total photon counts during 40 min of treatment. Values are mean ± SE (n = 20). (B) Activation profile of MAPKs in response to a time-course treatment with 1 μM flg22 or epiBL in 2-wk-old Col-0 seedlings. Arrowheads indicate phosphorylated MPK3 and MPK6. Blots stained with colloidal brilliant blue (CBB) are presented to show equal loading. (C) Quantitative RT-PCR analysis of FRK1 and At2g17740 expression in 2-wk old Col-0 seedlings treated with 100 nM flg22 or epiBL for 0, 1, or 3 h. Transcript levels are normalized to the U-box gene and are presented as relative to the value at 0 h. Values are mean ± SD (n = 3). (D) Growth of Pto DC3000 in Col-0 leaves pretreated with water, 1 μM flg22, or epiBL for 24 h and then syringe-infiltrated with 10⁵ cfu/mL of bacteria. Bacterial growth was determined 2 d after inoculation. Values are mean ± SE (n = 8; ***P < 0.001). Similar results were observed in at least two independent experiments.

Fig. 2.

Activation of BAK1 by PAMP perception does not modulate BR signaling. (A) BES1-GFP phosphorylation (detected as band shift) after the indicated times of seedling treatment with 1 μM epiBL and/or 1 μM flg22 by using anti-GFP antibodies. Blot stained with CBB is presented to show equal loading. (B) Phenotype of 2-wk-old Col-0, bri1-9, and bri1-301 Arabidopsis seedlings treated with buffer or different concentrations of flg22 or elf18 for 8 d. (Scale bar: 1 cm.) (C) Quantitative RT-PCR of CPD gene expression in 2-wk-old Col-0 seedlings grown with or without 10 nM flg22 for 1 wk and then treated with 1 μM epiBL for the indicated time. Transcript levels are normalized to the U-box gene and are presented as relative to mock treatment at time 0. Values are mean ± SD (n = 3; ***P < 0.001). Similar results were observed in at least three independent experiments.

Fig. 3.

EpiBL perception inhibits PTI signaling. (A) Effect of pretreatment with 1 μM epiBL for the indicated time on ROS triggered by 50 nM flg22 in Col-0 leaf discs. Total ROS production during 40 min of treatment is expressed as percentage of flg22-treated Col-0 without epiBL treatment. Values are mean ± SE (n = 20). (B). Quantitative RT-PCR of FRK1 and At2g17740 gene expression in 2-wk-old Col-0 seedlings treated with 1 μM flg22 and/or 1 μM epiBL for the indicated time. Transcript levels are normalized to the U-box gene and are presented as relative to time 0. Values are mean ± SD (n = 3). (C) Seedling growth inhibition induced by increasing concentrations of flg22 in absence (◇) or presence (◆) of 1 μM epiBL. Values are mean ± SD (n = 12; **P < 0.01 and ***P < 0.001). Similar results were observed in at least two independent experiments.

Fig. 4.

EpiBL perception does not affect the dynamic and the activity of the FLS2–BAK1–BIK1 complex. (A) IP of BAK1 in Col-0 seedlings treated with 1 μM flg22 (F) and/or epiBL (BL) for 10 min or 90 min (with or without 5 h epiBL pretreatment). Coimmunoprecipitated proteins were further analyzed by using anti-FLS2 or anti-BAK1 antibodies. (B) IP of GFP-tagged proteins from BRI1-eGFP or FLS2-3myc-GFP seedlings treated with 1 μM epiBL or flg22 (respectively) for 0, 10, and 90 min. Coimmunoprecipitated proteins were further analyzed by using anti-GFP or anti-BAK1 antibodies. (C) IP of BAK1-GFP proteins from transgenic seedlings treated as in A. Immunoblot is analyzed by using anti-BAK1 and anti-pThr antibodies. (D) IP of FLS2 or BAK1 from Col-0 seedlings treated with 1 μM flg22 (F) and/or epiBL (BL) for 10 min (with or without 90 min epiBL pretreatment). Immunoprecipitated proteins were then incubated in presence of radioactive [³²P]γ-ATP. Immunoblots are analyzed by using anti-FLS2 or anti-BAK1 antibodies. In vitro phosphorylation is revealed in the autoradiogram (i.e., ³²P). (E) BIK1 phosphorylation (detected as a band shift) in BIK1-HA seedlings treated as in A. The blots stained with CBB are presented to show equal loading. Molecular weights (in kDa) are indicated. Similar results were observed in at least two independent experiments.

Fig. 5.

EpiBL perception inhibits BAK1-independent chitin-mediated PTI signaling. (A) IP of BAK1 in Col-0 seedlings treated with water (“0”), 1 μM flg22 (“F”), or 1 mg/mL chitin (“C”) for the indicated time. Coimmunoprecipitated proteins were further analyzed by using anti-FLS2 or anti-CERK1 antibodies. The star indicates a nonspecific band. The blot stained with CBB is presented to show equal loading. (B) Oxidative burst in leaf discs from 5-wk old Col-0 and bak1-4 plants pretreated for 16 h with mock or 1 μM epiBL, then supplemented with 1 μM flg22, 1 mg/mL chitin, or mock. ROS production is presented as total photon counts during 40 min of treatment, and values are mean ± SE (n = 20; ***P < 0.001).