Flagellin induces innate immunity in nonhost interactions that is suppressed by Pseudomonas syringae effectors

Abstract

Arabidopsis NONHOST1 (NHO1) is required for limiting the in planta growth of nonhost Pseudomonas bacteria but completely ineffective against the virulent bacterium Pseudomonas syringae pv. tomato DC3000. However, the molecular basis underlying this observation remains unknown. Here we show that NHO1 is transcriptionally activated by flagellin. The nonhost bacterium P. syringae pv. tabaci lacking flagellin is unable to induce NHO1, multiplies much better than does the wild-type bacterium, and causes disease symptoms on Arabidopsis. DC3000 also possesses flagellin that is potent in NHO1 induction, but this induction is rapidly suppressed by DC3000 in a type III secretion system-dependent manner. Direct expression of DC3000 effectors in protoplasts indicated that at least nine effectors, HopS1, HopAI1, HopAF1, HopT1-1, HopT1-2, HopAA1-1, HopF2, HopC1, and AvrPto, are capable of suppressing the flagellin-induced NHO1 expression. One of the effectors, HopAI1, is conserved in both animal and plant bacteria. When expressed in transgenic Arabidopsis plants, HopAI1 promotes growth of the nonpathogenic hrpL- mutant bacteria. In addition, the purified phytotoxin coronatine, a known virulence factor of P. syringae, suppresses the flagellin-induced NHO1 transcription. These results demonstrate that flagellin-induced defenses play an important role in nonhost resistance. A remarkable number of DC3000 virulence factors act in the plant cell by suppressing the species level defenses, and that contributes to the specialization of DC3000 on Arabidopsis.

Fig. 1.

NHO1 is transcriptionally induced by nonhost bacteria and flagellin. (a) A luciferase image of NHO1-LUC transgenic leaves inoculated with water, nonhost strain Pph, or virulent strain DC3000 for 24 h. (b) Time course of NHO1-LUC expression in plants inoculated with water, Pph, or DC3000 bacteria. (c) NHO1-LUC activity of plants inoculated with 1 μM flg22^P.aeruginosa or flg22^{A.tumefaciens} at the indicated hours. The experiments were repeated numerous times with similar results.

Fig. 2.

Flagellin is required for NHO1 induction and resistance to Ptab. (a) NHO1-LUC activity of plants inoculated with the wild-type and fliC^- mutant strains of Ptab. (b) Disease symptoms of Arabidopsis plants (Col-0) 7 days after inoculation with the wild-type (WT) and fliC^- mutant strains of Ptab (10⁶ cfu/ml). (c) Bacterial growth of the wild-type (WT) and fliC^- mutant Ptab strains on Arabidopsis plants (Col-0). The experiments were repeated three times with similar results.

Fig. 3.

DC3000 flagellin transiently induces NHO1 and fails to confer disease resistance. (a) NHO1-LUC expression in plants inoculated with water, the wild-type (WT) or fliC^- mutant DC3000 strains at the indicated hours after inoculation. (b) Bacterial growth assay of Col-0 plants infiltrated with the wild-type (WT) or fliC^- mutant strains of DC3000. The experiments were repeated three times with similar results.

Fig. 4.

DC3000 requires type III effectors to suppress NHO1 expression. (a) NHO1-LUC plants were inoculated with the wild-type (WT), hrpA^-, hrcC^-, or hrpL^- mutant DC3000 strains, and relative luciferase activity was measured 0, 12, and 24 h after inoculation. (b) Kinetics of NHO1-LUC expression in response to the wild-type (WT) and hrpL^- mutant DC3000 strains. (c) Expression of DC3000 effectors blocks flg22-induced NHO1-LUC expression. Protoplasts were transfected either with the empty vector (V) or the indicated effector constructs, and relative LUC activity was measured 12 h after addition of flg22. Vector-transfected protoplasts treated with ddH₂O were used as a control for basal NHO1-LUC expression (V-). Each data point consists of three replicates. The error bar represents standard error. The experiments were repeated three times with similar results.

Fig. 5.

HopAI1 promotes virulence in plants. (a) hopAI1 expression induces chlorosis. Transgenic hopAI1-FLAG (line 2) and wild-type (WT) plants were sprayed with 50 μM estradiol and photographed 5 days later. (b) hopAI1 expression enhances bacterial growth in plants. Transgenic hopAI1-FLAG (line 2) and wild-type (WT) plants were sprayed with either buffer or 50 μM estradiol 1 day before inoculation with the hrpL^- mutant. Bacteria population in the leaf was determined at the indicated times. Error bars indicate standard error.

Fig. 6.

Coronatine partially suppresses NHO1 expression. (a) Purified coronatine inhibits the flagellin-induced NHO1-LUC expression. NHO1-LUC activity of plants infiltrated with water, 1 μM flg22^P.s.tabaci alone, 1 μM flg22^P.s.tabaci plus 200 ng/ml purified coronatine, or 1 μM flg22^{A.tumefaciens}. (b) Purified coronatine inhibits the Pph-induced NHO1-LUC expression. NHO1-LUC activity of plants inoculated with water, Pph, or Pph plus 200 ng/ml coronatine (cronatine+Pph). The experiments were repeated twice with similar results.