Ethylene signaling regulates accumulation of the FLS2 receptor and is required for the oxidative burst contributing to plant immunity

Abstract

Reactive oxygen species (ROS) are potent signal molecules rapidly generated in response to stress. Detection of pathogen-associated molecular patterns induces a transient apoplastic ROS through the function of the NADPH respiratory burst oxidase homologs D (RbohD). However, little is known about the regulation of pathogen-associated molecular pattern-elicited ROS or its role in plant immunity. We investigated ROS production triggered by bacterial flagellin (flg22) in Arabidopsis (Arabidopsis thaliana). The oxidative burst was diminished in ethylene-insensitive mutants. Flagellin Sensitive2 (FLS2) accumulation was reduced in etr1 and ein2, indicating a requirement of ethylene signaling for FLS2 expression. Multiplication of virulent bacteria was enhanced in Arabidopsis lines displaying altered ROS production at early but not late stages of infection, suggesting an impairment of preinvasive immunity. Stomatal closure, a mechanism used to reduce bacterial entry into plant tissues, was abolished in etr1, ein2, and rbohD mutants. These results point to the importance of flg22-triggered ROS at an early stage of the plant immune response.

Figure 1.

Flg22-stimulated oxidative burst and FLS2 abundance in ethylene-insensitive mutants. A, Flg22-triggered ROS production was monitored in liquid-grown intact seedlings of the indicated genotypes over time. Depicted are average values (n = 18); error bars represent ±sd. Similar results were obtained in multiple independent experiments. Letters indicate significant differences at P < 0.05. B, Immunoblot analysis of the indicated genotypes using specific anti-FLS2 and anti-BAK1 antibodies. Coomassie Brilliant Blue staining is shown for equal loading. Several independent experiments revealed similar results.

Figure 2.

FLS2 expression levels in ethylene-insensitive mutants. Quantitative real-time PCR monitoring of FLS2, BAK1, and RbohD transcript levels in the indicated genotypes is shown. Tubulin was used as a control. Depicted are average values of two independent experiments (n = 6); error bars represent ±sd. Letters indicate significant differences at P < 0.05.

Figure 3.

Flg22-stimulated early and late responses in ethylene-insensitive mutants. A, Seedling growth of the indicated genotypes was measured in the absence or presence of 100 nm flg22. Error bars represent ±sd. Letters indicate significant differences at P < 0.05. Similar results were obtained in three independent experiments. B, Flg22-induced MPK6 and MPK3 activation was determined by in-gel MAP kinase assays at the indicated time points and seedling genotypes. Coomassie Brilliant Blue staining is shown for equal loading.

Figure 4.

Flg22-induced oxidative burst in leaf discs. A, Mature leaves of the indicated genotypes were excised into discs of approximately equal sizes and monitored for flg22-mediated ROS production. Depicted are average values (n = 8); error bars represent ±sd. RLU, Relative light unit. Letters indicate significant differences at P < 0.05. B, FLS2 immunoblot analysis. Coomassie Brilliant Blue staining is shown for equal loading.

Figure 5.

Bacterial growth in ethylene-insensitive mutants. Plants of the indicated genotypes were surface inoculated with PtoDC3000 ΔAvrPto/ΔAvrPtoB, and bacterial multiplication was monitored at 4, 24, and 72 h post infection (hpi). Shown are average values of three independent experiments (n = 16); error bars represent ±sd. Letters indicate significant differences at P < 0.05.

Figure 6.

Flg22-triggered stomatal closure. Leaves of the indicated seedlings were untreated, stimulated with flg22, or infected with PtoDC3000 ΔAvrPto/ΔAvrPtoB, and stomatal aperture was measured microscopically as width to length ratio. Depicted are average values; error bars represent ±sd (n = 140 stomata of three independent experiments). Letters indicate significant differences at P < 0.1 (b) or P < 0.05 (c).

Figure 7.

Bacterial growth in plants with altered ROS accumulation. Plants of the indicated genotypes were surface infected with PtoDC3000 ΔAvrPto/ΔAvrPtoB, and bacterial multiplication was determined at 4, 24, and 72 h post infection (hpi). Shown are average values of three independent experiments (n = 16); error bars represent ±sd. Letters indicate significant differences at P < 0.05.