The Arabidopsis flagellin receptor FLS2 mediates the perception of Xanthomonas Ax21 secreted peptides - PubMed (original) (raw)

The Arabidopsis flagellin receptor FLS2 mediates the perception of Xanthomonas Ax21 secreted peptides

Cristian H Danna et al. Proc Natl Acad Sci U S A. 2011.

Abstract

Detection of microbes by plants relies in part on an array of pattern-recognition receptors that recognize conserved microbial signatures, so-called "microbe-associated molecular patterns." The Arabidopsis thaliana receptor-like kinase FLS2 is the pattern-recognition receptor for bacterial flagellin. Similarly to FLS2, the rice transmembrane protein XA21 is the receptor for the sulfated form of the Xanthomonas oryzae pv. oryzae secreted protein Ax21. Here we show that Ax21-derived peptides activate Arabidopsis immunity, triggering responses similar to those elicited by flagellin, including an oxidative burst, induction of defense-response genes, and enhanced resistance to bacterial pathogens. To identify Arabidopsis Xa21 functional homologs, we used a reverse genetics approach to screen T-DNA insertion mutants corresponding to all 47 of the Arabidopsis genes encoding non-RD kinases belonging to the interleukin-1 receptor-associated kinase (IRAK) family. Surprisingly, among all of these mutant lines, only fls2 mutants exhibited a significant loss of response to Ax21-derived peptides. Ax21 peptides also failed to activate defense-related responses in an fls2-24 mutant that does not bind Flg22. Moreover, a Flg22Δ2 variant of Flg22 that binds to FLS2 but does not activate FLS2-mediated signaling suppressed Ax21-derived peptide signaling, indicating mutually exclusive perception of Flg22 or Ax21 peptides by FLS2. The data indicate that FLS2 functions beyond flagellin perception to detect other microbe-associated molecular patterns.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Ax21-derived peptides trigger MAMP-responsive gene expression in Arabidopsis. Leaves from 6-wk-old Col-0 transgenic plants carrying WRKY11::GUS or MYB51::GUS constructs were infiltrated with 1 μM peptides (Right) or water (Left) and stained with GUS, as described in

SI Methods

Fig. 2.

Ax21-derived peptides trigger a hydrogen peroxide burst. Ten-day-old Col-0 wild-type seedlings were grown in 96-well plates and mock-treated or elicited with axY22, Flg22, or Elf26 (A) or with axYs22 or axY22A (B) at the indicated concentrations: axYs22 is the sulfated 17-mer that is active in rice; axY22 is the nonsulfated version of axYs22; axY22A contains a Tyr→Ala substitution. Neither axY22 nor axY22A can trigger rice XA21-mediated immunity. Each point represents the mean of six seedlings. Error bars represent ± SD of the mean. Essentially identical results were obtained in at least three independent experiments.

Fig. 3.

Ax21-derived peptides trigger enhanced resistance against bacteria. (A) Ten-d-old seedlings were grown in 12-well plates, elicited with 1 μM Flg22 or 1 μM, 10 μM, or 100 μM axY22, axYs22, or axY22A for 24 h, and then infected with Psm-LUX. Bacterial titer was assessed as CFU/mg fresh weight seedling tissue 36 h after inoculation: axYs22 is the sulfated 17-mer that is active in rice; axY22 is the nonsulfated version of axYs22; axY22A contains a Tyr→Ala substitution. Neither axY22 nor axY22A can trigger rice XA21-mediated immunity. (B) Seedlings were elicited with 100 μM axYs22 for 24 h and then inoculated with Xcc. Each column represents the bacterial titer 24 h after inoculation as CFU/mg fresh weight seedling tissue and is the mean of three wells containing 20 seedlings each. Error bars represent ± SD of the mean. Essentially identical results were obtained in at least three independent experiments. *P < 0.001 compared with mock (t test).

Fig. 4.

FLS2 is necessary for Ax21-derived peptide perception. (A) A hydrogen peroxide burst was elicited in 10-d-old Col-0 wild-type or fls2 mutant (SAIL_691_C04) seedlings in 96-well plates with axYs22-A1 (10 μM) or Flg22 (1 μM). Each datapoint represents the mean of six seedlings (six wells). Error bars represent ± SD of the mean. (B) Ten-d-old Col-0 wild-type or fls2 (SAIL_691_C04) seedlings were grown in 96-well plates, elicited with axYs22-A1 (10 μM) or Flg22 (1 μM) for 24 h and then infected with _Psm_-LUX. Bacterial titer was determined with a 96-well plate scintillation counter each hour between 20 and 24 h after inoculation. Each datapoint represents the mean of six seedlings (six wells). Error bars represent ± SD of the mean. (C) Ten-day-old Col-0 wild-type or fls2 mutant (SAIL_691_C04) seedlings were grown in 12-well plates and elicited with various Ax21 peptides and infected with Psm_-LUX as in Fig. 3_A. A second fls2 insertion mutant (Salk_121477) showed identical results. Each column represents the mean of three wells containing 20 seedlings each. Error bars represent ± SD of the mean. Essentially identical results were obtained in at least three independent experiments. *P < 0.001 compared with mock (t test).

Fig. 5.

FLS2-mediated perception of Ax21-derived peptides mimics Flg22 perception. (A) A hydrogen peroxide burst was elicited in 10-d-old Col-0 wild-type or AtrbohD mutant seedlings with axYs22-A1 (10 μM) or Flg22 (1 μM) in 96-well plates. Each datapoint represents the mean of six seedlings (six wells). Error bars represent ± SD of the mean. (B) Ten-d-old Col-0 wild-type, fls2 mutant (SAIL_691_C04), or bak1 mutant (SALK_116202) seedlings were grown in 12-well plates and elicited with axYs22-A1 (10 μM) or Flg22 (1 μM) and then infected with Psm_-LUX as in Fig. 3_A. Columns represent the mean of three wells containing 20 seedlings each. Error bars represent ± SD of the mean. (C) Ten-day-old Col-0 wild-type or fls2 mutant seedlings were grown in 12-well plates and elicited with Flg22 (1 μM) or axYs22-A1 (5 μM) for 3 h. RNA was extracted and qRT-PCR analysis was carried out as described in

SI Methods

. Gene expression is shown as fold-change compared with mock treatment. Columns represent the mean of three independent qRT-PCR reactions. Error bars represent ± SEM. Essentially identical results were obtained in at least three independent experiments. *P < 0.001 and **P < 0.01 compared with mock (t test).

Fig. 6.

The Flg22 binding domain of FLS2 is also required for Ax21-derived peptide perception. (A) A hydrogen peroxide burst was monitored in 10-d-old L_er_ wild-type or fls2-24 mutant seedlings in 96-well plates after elicitation with Flg22 (1 μM) or axYs22-A1 (10 μM). (B) A hydrogen peroxide burst was monitored in Col-0 wild-type seedlings after elicitation with axYs22-A1, Flg22Δ2, or a combination of both peptides. Each datapoint represents the mean of six seedlings (six wells). Error bars represent ± SD of the mean. Essentially identical results were obtained in at least three independent experiments (A) or two independent experiments (B).

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The Arabidopsis flagellin receptor FLS2 mediates the perception of Xanthomonas Ax21 secreted peptides - PubMed (original) (raw)