Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response

Abstract

Reactive oxygen intermediates (ROI) are strongly associated with plant defense responses. The origin of these ROI has been controversial. Arabidopsis respiratory burst oxidase homologues (rboh genes) have been proposed to play a role in ROI generation. We analyzed lines carrying dSpm insertions in the highly expressed AtrbohD and AtrbohF genes. Both are required for full ROI production observed during incompatible interactions with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000(avrRpm1) and the oomycete parasite Peronospora parasitica. We also observed reduced cell death, visualized by trypan blue stain and reduced electrolyte leakage, in the Atrboh mutants after DC3000(avrRpm1) inoculation. However, enhanced cell death is observed after infection of mutant lines with P. parasitica. Paradoxically, although atrbohD mutation eliminated the majority of total ROI production, atrbohF mutation exhibited the strongest effect on cell death.

Figure 1

Arabidopsis atrbohD and atrbohF mutants. (A) Schematic representation of the transposon insertions in the Atrboh D and F genes. Boxes represent the exons of the genes, and red triangles mark the dSpm transposon insertions. (B) Representative 4-week-old rosettes. (C) Trypan blue staining of an atrbohD/F double mutant leaf, displaying spontaneous necrosis. (D) Detail of an atrbohD/F leaf stained with aniline blue to show callose deposition. Note that infection experiments are performed on young plants that do not exhibit ectopic cell death. [Bar = 1 cm (B) and 1 mm (D).]

Figure 2

Reduced ROI accumulation and cell death in the atrboh mutants after inoculation of avirulent bacteria. (A) In situ detection of peroxides by using DAB staining on wt Col-0 and atrboh mutant leaves 5 h postinoculation with DC3000(avrRpm1) at 2.5 × 10⁷ cfu/ml. (B) Quantitative analysis of DAB on leaves 6 h postinoculation with DC3000(avrRpm1) at 2.5 × 10⁷ cfu/ml, DC3000 (same inoculum concentration), or 10 mM MgCl₂. (Bars = SD.) (C) Detail of leaves stained with DAB 5 h postinoculation with DC3000(avrRpm1) at 2.5 × 10⁷ cfu/ml. Genotypes are labeled at the top, left to right: Col-0, atrbohD, atrbohF, and atrbohD/F. (D) Detail of leaves stained with trypan blue 5 h postinoculation with DC3000(avrRpm1) at 2.5 × 10⁷ cfu/ml. (E) Detail of leaves stained with trypan blue 12 h postinoculation with lower dose of DC3000(avrRpm1), 10⁶ cfu/ml. (Bar = 25 μm.) All images in C–E have the same magnification.

Figure 3

Reduced electrolyte leakage in atrboh mutants after inoculation with avirulent bacteria. (A) Conductivity (μS/cm) of solution containing leaf discs from either wt Col-0 or atrbohD/F mutant inoculated with avirulent bacteria DC3000(avrRpm1) at 10⁷ cfu/ml, virulent bacteria DC3000 at 10⁷ cfu/ml, or 10 mM MgCl₂. (B) Detailed differences in conductivity between Col-0 and the atrboh mutants during the incompatible interaction DC3000(avrRpm1) at 10⁷ cfu/ml. Each value represents the mean and SD of three replicates (compatible interaction and MgCl₂) or four replicates (incompatible interactions) per experiment. The experiment was repeated three times with similar results.

Figure 4

Reduced peroxide accumulation and enhanced cell death in the atrboh mutants after inoculation with Pp isolate Emco5. (A) DAB staining of leaves 3 days after inoculation with Pp isolate Emco5. Arrows indicate HR sites in atrbohD/F. (B) Trypan blue stain of leaves from the same experiment 3 days after inoculation with Pp isolate Emco5. The experiment was repeated four times; ≈10 leaves per genotype per experiment analyzed. (Bar = 50 μm for all.)

Figure 5

Reduced sporangiophore formation in the atrboh mutants. The histogram represents the percentage of first leaves of seedlings (genotypes at bottom) that display >20, 11–20, 1–10, or 0 sporangiophores 7 days postinoculation with Pp isolate Emco5. The data are the mean and SD from three different experiments representing more than 200 total leaves evaluated for each genotype.