Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance

Abstract

The oxidative burst is an early response to pathogen attack leading to the production of reactive oxygen species (ROS) including hydrogen peroxide. Two major mechanisms involving either NADPH oxidases or peroxidases that may exist singly or in combination in different plant species have been proposed for the generation of ROS. We identified an Arabidopsis thaliana azide-sensitive but diphenylene iodonium-insensitive apoplastic oxidative burst that generates H(2)O(2) in response to a Fusarium oxysporum cell-wall preparation. Transgenic Arabidopsis plants expressing an anti-sense cDNA encoding a type III peroxidase, French bean peroxidase type 1 (FBP1) exhibited an impaired oxidative burst and were more susceptible than wild-type plants to both fungal and bacterial pathogens. Transcriptional profiling and RT-PCR analysis showed that the anti-sense (FBP1) transgenic plants had reduced levels of specific peroxidase-encoding mRNAs, including mRNAs corresponding to Arabidopsis genes At3g49120 (AtPCb) and At3g49110 (AtPCa) that encode two class III peroxidases with a high degree of homology to FBP1. These data indicate that peroxidases play a significant role in generating H(2)O(2) during the Arabidopsis defense response and in conferring resistance to a wide range of pathogens.

Figure 1. Elicitation of an oxidative burst in Arabidopsis tissue cultures

(a) Comparative level of the oxidative burst in Arabidopsis cell-suspension cultures treated with 100 µg ml ⁻¹ glucose equivalents of Fusarium oxysporum elicitor in the absence (●) or presence (○) of 10 U catalase. Also shown is the burst in response to 25 µg ml⁻¹ Colletotrichum lindemuthianum elicitor (■), the optimum for reactive oxygen species (ROS) production in French bean cells. Production of ROS was measured by the xylenol orange assay as described in Experimental procedures. (b) Dose-response curve for inhibition of ROS production in F. oxysporum elicitor-treated Arabidopsis cell-suspension cultures by sodium azide. (c) Comparative dose-response curve for diphenylene iodonium (DPI) inhibition of the oxidative burst in Arabidopsis cell-suspension cultures treated with F. oxysporum elicitor (○) or French bean cells treated with C. lindemuthianum elicitor (□) using the xylenol orange method. Values are means ± SD from replicates from at least five independent experiments.

Figure 2. Elicitation of reactive oxygen species (ROS) in Arabidopsis leaves

(a) Production of ROS in leaf discs of wild-type Col-0 (●) or FBP1 transgenic lines 1.1 (◇) or 1.2 (△) treated with 25 µg ml⁻¹ Fusarium oxysporum elicitor. (b) In situ detection of H₂O₂ by 3,3′-diaminobenzidine (DAB) staining. Leaves of non-transformed Col-0 plants or homozygous transgenic anti-sense FBP1 H₄ plants were inoculated with Pto DC3000(avrRpm1) at OD₆₀₀ = 2.0 (10⁷ CFU cm⁻²), detached after 2 h and infiltrated under gentle vacuum with 1 mg ml⁻¹ DAB containing 0.05% v/v Tween 20 and 10 mm sodium phosphate buffer pH 7.0. The reaction was terminated at 6–7 h post-inoculation when a brown precipitate began to be visible in Col-0 leaves. Leaves were examined after bleaching by light microscopy (100× magnification). (c) In situ detection of H₂O₂ by cerium chloride staining. Leaves of non-transformed Col-0 plants or homozygous transgenic FBP1 H₄ plants were infiltrated with Pto DC3000(avrRpm1) at 10⁷ CFU cm⁻² followed by CeCl₃ staining and electron microscope detection. Scale bar, 0.5 µm. Ps, Pseudomonas syringae; CW, cell wall.

Figure 3. Susceptibility of FBP1 transgenic plants to bacterial and fungal pathogens

(a) Growth of Pto DC3000 (black bars) or Psm ES4326 (open bars) 3 days post-inoculation of wild-type Col-0 or homozygous FBP1 transgenic H₄ leaves at a dose of 10⁴ CFU cm⁻² leaf area. Means ± SE of six determinations are shown. (b) Growth of virulent (ES4326, black bars) or avirulent [ES4326(avrRpt2), open bars] Psm strains 3 days post-inoculation of wild-type Col-0 or homozygous FBP1 transgenic H₄ leaves with 10³ CFU cm⁻² leaf area. Means ± SE of eight determinations are shown. Differences between Col and H₄ were statistically significant according to a t-test, with P ≤ 0.001. The experiment was repeated twice with similar results. (c) Leaf pairs of Col-0 (left) and FBP1 transgenic line H₄ plants (right) either mock-inoculated or inoculated with virulent spores of Botrytis cinerea. (d) Opportunistic infection of FBP1 transgenic line H₄ by the powdery mildew pathogen Golovinomyces orontii. Wild-type Col-0 and FBP1 transgenic plants were grown side-by-side in a flat (left panel) in a glasshouse contaminated with G. orontii spores. The FBP1 transgenic plants, but not the wild-type plants, develop symptoms of G. orontii infection. The two right-hand panels show close-ups of wild-type and FBP1 plants.

Figure 4. Susceptibility of FBP1 transgenic lines to the fungal toxin fumonisin B1

Top row, Col-0 leaf pairs (left) compared with leaf pairs of FBP1 transgenic line H₄ (right) mock-infiltrated with 0.14% methanol or infiltrated with 10 µM FB1. Bottom row, non-infiltrated leaves from plants treated with 0.14% methanol or 10 µm FB1, respectively.

Figure 5. Peroxidases in leaves of FBP1 transgenic lines

(a) Relative specific activities of peroxidases in soluble (■) and bound (□) fractions were determined for Col-0 and FBP1 transgenic line H₄ extracts using ABTS as substrate, as described in Experimental procedures. The experiment was repeated three times with similar results. (b) Isoelectric focusing profiling of bound peroxidases from Col-0 and FBP1 transgenic line H₄. Equal amounts (20 µg) of protein were loaded per track and the zymogram was developed using o-danisodine as described in Experimental procedures.

Figure 6. Transcript levels of the peroxidase-encoding genes AtPCa (At3g49110) and AtPCb (At3g49120) in Col-0 and FBP1 transgenic line H₄

(a) Ratio of At3g49110 expression in H₄ versus wild-type Col-0 in plants that were either mock-inoculated or inoculated with Pto DC3000(avrRpm1). Leaves were harvested 5 h post-inoculation. Values are averages of three independent experiments. (b) Ratio of At3g49120 expression in H₄ versus wild-type Col-0 in plants that were either mock-inoculated or inoculated with Pto DC3000(avrRpm1). (c) Change in transcript level of At3g49110 and At3g49120 in wild-type Col-0 in response to inoculation with Pto DC3000(avrRpm1). Fold-change is relative to mock-inoculated. Steady-state mRNA in 4-week old Col-0 and H₄ was assayed by real-time RT-PCR and results from three independent biological replicates were averaged.