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Nonlegumes Respond to Rhizobial Nod Factors by Suppressing the Innate Immune Response

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Science  20 Sep 2013:
Vol. 341, Issue 6152, pp. 1384-1387
DOI: 10.1126/science.1242736

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  1. Fig. 1 Nod factor (NF) and chitotetraose (C4) treatment reduces flg22-triggered immunity in soybean leaves.

    (A) Nod factor and chitotetraose reduced flg22-triggered ROS production. Leaf discs were treated with H2O, Nod factor (100 nM), chitotetraose (10 μM), and flg22 (100 nM) with or without the addition of Nod factor or chitotetraose. (B) Pretreatment enhanced the suppressive effect of C4 on flg22-triggered ROS production. (C) Nod factor (1 nM) reduced flg22-triggered ROS production. Leaf discs were pretreated with different concentrations of Nod factor for 30 min before flg22 (100 nM) treatment. (A to C) The data are shown as means ± SD (error bars; n = 6 leaf discs). Asterisks indicate significant differences from flg22 treatment (**P ≤ 0.01, *P ≤ 0.05). All experiments were repeated twice with similar results. (D) Nod factor and chitotetraose reduced flg22-induced MAP kinase (MAPK) phosphorylation. Lower panels shows representative Western blot; upper panel shows quantification of normalized protein levels to flg22 treatment. Data are means ± SE (error bars) from three independent biological repeats.

  2. Fig. 2 Nod factor and chitotetraose treatment reduces flg22-triggered immunity in Arabidopsis.

    (A) Nod factor and chitotetraose reduced flg22-triggered ROS production in Arabidopsis leaves. (B) Nod factor and chitotetraose reduced flg22-triggered callose deposition. Representative images are shown here, and the quantification data are shown in fig. S5. Scale bar, 1 mm. DMSO, dimethyl sulfoxide. (C) Nod factor and chitotetraose reduced flg22-triggered bacterial growth restriction. Five-week-old Arabidopsis plants were infiltrated with the indicated treatment before Pseudomonas syringae pv. tomato DC3000 inoculation at a concentration of 105 colony-forming units (CFU)/ml. Bacterial growth was determined 3 days after inoculation. The data are shown as means ± SD (error bars) from three replicates (*P ≤ 0.05). (D) Nod factor and chitotetraose reduced flg22-triggered calcium influx in Arabidopsis seedlings. Leaf disc (A) or 6-day-old seedlings (D) were pretreated with H2O, Nod factor (100 nM), and chitotetraose (10 μM) for 1 hour before flg22 (10 nM) treatment. Data are means ± SD (error bars; n = 6 leaf discs; **P ≤ 0.01, *P ≤ 0.05). All experiments were repeated twice with similar results.

  3. Fig. 3 Nod factor induces FLS2 protein degradation in Arabidopsis.

    (A) Nod factor induces FLS2 protein degradation, and the protein degradation was inhibited by the addition of MG132. Ten-day-old seedlings were treated with or without MG132 (50 μM) for 1 hour before Nod factor (100 nM) treatment. Total protein was extracted 1 hour after Nod factor treatment. (B) Nod factor–induced FLS2 protein degradation was absent in bak1-4 mutants. Immunoblot analysis was performed with the anti-FLS2 antibody. Ponceau S staining was used for protein loading control. (A and B) Lower panels show representative Western blot; upper panels show quantification of normalized FLS2 levels to the control. The data are shown as means ± SE (error bars) from three independent biological repeats. (C) Nod factor addition results in the reduced level of FLS2:GFP from the plasma membrane. Five-day-old FLS2::FLS2:GFP seedlings were treated with or without Nod factor (100 nM). Images were taken from cotyledon 1 hour after Nod factor treatment. Costaining with FM4-64 (10 μM) highlights the plasma membrane. Scale bars, 10 μm.

  4. Fig. 4 AtLYK3 is required for Nod factor suppression of flg22-triggered immunity.

    (A) lyk3 mutants are defective in Nod factor suppression of flg22-triggered ROS production. (B) Transgenic plants ectopically overexpressing LYK3 (LYK3-OX) are hypersensitive to Nod factor suppression of flg22-triggered ROS production. Leaf discs in (A) and (B) were pretreated with H2O or Nod factor (100 nM) for 1 hour before flg22 (10 nM) treatment. The relative ROS production was calculated as a percentage of the value of flg22 plus Nod factor divided by the value of flg22. The data are shown as means ± SD (error bars; n = 6 leaf discs). Asterisks indicate significant difference from the wild type (**P ≤ 0.01, *P ≤ 0.05). All experiments were repeated twice with similar results. (C) lyk3 mutants and LYK3-OX transgenic plants exhibited altered response to Nod factor suppression of flg22-triggered MAP kinase phosphorylation. Lower panels show representative Western blot; upper panel shows quantification of normalized protein levels to flg22 treatment. The data are shown as means ± SE (error bars) from four independent biological repeats.