Genetic elucidation of nitric oxide signaling in incompatible plant-pathogen interactions

Abstract

Recent experiments indicate that nitric oxide (NO) plays a pivotal role in disease resistance and several other physiological processes in plants. However, most of the current information about the function of NO in plants is based on pharmacological studies, and additional approaches are therefore required to ascertain the role of NO as an important signaling molecule in plants. We have expressed a bacterial nitric oxide dioxygenase (NOD) in Arabidopsis plants and/or avirulent Pseudomonas syringae pv tomato to study incompatible plant-pathogen interactions impaired in NO signaling. NOD expression in transgenic Arabidopsis resulted in decreased NO levels in planta and attenuated a pathogen-induced NO burst. Moreover, NOD expression in plant cells had very similar effects on plant defenses compared to NOD expression in avirulent Pseudomonas. The defense responses most affected by NO reduction during the incompatible interaction were decreased H(2)O(2) levels during the oxidative burst and a blockage of Phe ammonia lyase expression, the key enzyme in the general phenylpropanoid pathway. Expression of the NOD furthermore blocked UV light-induced Phe ammonia lyase and chalcone synthase gene expression, indicating a general signaling function of NO in the activation of the phenylpropanoid pathway. NO possibly functions in incompatible plant-pathogen interactions by inhibiting the plant antioxidative machinery, and thereby ensuring locally prolonged H(2)O(2) levels. Additionally, albeit to a lesser extent, we observed decreases in salicylic acid production, a diminished development of hypersensitive cell death, and a delay in pathogenesis-related protein 1 expression during these NO-deficient plant-pathogen interactions. Therefore, this genetic approach confirms that NO is an important regulatory component in the signaling network of plant defense responses.

Figure 1.

Expression of E. coli hmp in transgenic Arabidopsis as a functional NOD. A, Northern-blot analysis illustrating time-dependent accumulation of hmp transcripts after treatment of hmp8 plants with 3 μm DEX (c, no DEX). B, Western blot demonstrating the appearance of correctly sized Hmp protein in leaf extracts; time course as in A. C and D, Electrochemically measured degradation kinetics of 10 μm NO in leaf extracts of wild-type and hmp8 transgenic Arabidopsis. Error bars represent the sds of five independent measurements. C, Hmp8 plants without DEX-induced transgene expression. D, Wild-type and hmp8 plants 1 d after DEX treatment. NO concentrations of less than 1 μm were reached for wild-type, noninduced hmp8, and DEX-induced hmp8 plants at 197 s, 202 s, and 131 s, respectively. E, NO emission from intact wild-type and hmp8 plants 1 d after DEX treatment. Plants were first incubated in the dark for 10 min and then illuminated for 60 min to cause nitrate reductase-dependent NO emission. The light was switched off again, which gave rise to a characteristic light-off peak (see “Results”). Experiments were repeated three times with similar results.

Figure 2.

Pathogen-induced DAF2-DA fluorescence as a measure for NO production in DEX-treated wild-type and hmp8 plants. Leaves were pretreated with Pst (avrB) or MgCl₂ for 3 h and subsequently infiltrated with 10 μm DAF2-DA or control buffer (10 mm Tris/KCl, pH 7.2). Infiltrated leaf areas were analyzed 1 h later by confocal laser scanning microscopy. DAF2-DA fluorescence (green) was recorded using a channel with a 505- to 530-nm band-pass filter, and autofluorescence of chloroplast (red) was captured with a channel equipped with a 560-nm long-pass filter. A, Treatment of a wild-type Arabidopsis leaf with Pst (avrB) and control buffer. B, Wild-type Arabidopsis-MgCl₂ and DAF2-DA. C, Wild-type Arabidopsis-Pst (avrB) and DAF2-DA. D, Hmp8-Pst (avrB) and DAF2-DA. Seven independent samples were recorded for each condition, and representative leaf areas are shown.

Figure 3.

DAB staining of Arabidopsis leaves to assess H₂O₂ accumulation during the oxidative burst in DEX-treated wild-type and hmp8 transgenic plants. Solutions of Pst (avrB) were infiltrated into Arabidopsis leaves, and DAB staining was initiated 4 h after infection. A, Staining patterns of representative, MgCl₂-infiltrated wild-type or hmp8 leaves (control), Pst (avrB)-infected wild-type Arabidopsis leaves (Wt-avr), Pst (avrB)-infected hmp8 plants (hmp8-avr), and Pst (avrB/hmpX)-infected hmp8 plants (hmp8-avr/hmpX) 4 h after the respective treatment (100-fold magnification). B, Quantification of DAB staining in Pst (avrB)-infected wild-type and hmp8 leaves. The percentage of stained pixels inside the infiltration area was assessed as described in “Materials and Methods.” Values are shown as the mean ± sd of at least five leaves from different plants. Experiments were repeated three times with similar results.

Figure 4.

DAB staining of Arabidopsis leaves to assess their capability to degrade H₂O₂ in DEX-treated wild-type and hmp transgenic plants. Solutions of 2.5 mm Glc/2.5 units mL⁻¹ Glc oxidase were infiltrated into leaves and DAB staining was performed 1 h after infiltration. A, Staining patterns of representative leaves inside the infiltrated area (100-fold magnification). B, Percentage of stained pixels inside the infiltration zone. Values are shown as the mean ± sd of at least five leaves from different plants. Experiments were repeated three times with similar results.

Figure 5.

Expression of defense and cellular protectant genes in wild-type and hmp8 transgenic plants after Pst (avrB) challenge. Three parallel leaf samples were collected at the indicated times after infection for RNA extraction and northern-blot analysis. MgCl₂ (m)-infiltrated leaves were collected 4 h after infection. Experiments were repeated three times with similar results.

Figure 6.

Expression of PAL and CHS in DEX pretreated wild-type and hmp8 plants after UV exposure. Three parallel leaf samples were collected at the indicated times after the beginning of UV treatment. Experiments were repeated three times with similar results.

Figure 7.

Macroscopic HR symptoms 2 d after infiltration of DEX-pretreated wild-type and hmp8 plants with avirulent Pseudomonas. Bacteria were infiltrated into the left side of leaves. Seven parallels are shown for each condition. A, Wild-type plants-Pst (avrB). B, hmp8 plants-Pst (avrB). C, hmp8 plants-Pst (avrB/hmpX).

Figure 8.

Microscopic cell death after pathogen challenge of DEX-pretreated wild-type and hmp8 plants. Trypan blue staining was performed 24 h after infection. A, Staining patterns of representative leaves inside the infiltrated area (100-fold magnification). B, Percentage of stained pixels inside the infiltration area. Values are shown as the mean ± sd of at least six leaves from different plants. Experiments were repeated three times with similar results.

Figure 9.

SA contents of wild-type and hmp8 transgenic plants after challenge with avirulent Pst (± hmpX). Leaf samples were collected 8 h postinfection. Leaves were pretreated with DEX for 16 h. Bars indicated mean values of three independent measurements. Control, MgCl₂-infiltrated plants. A, Free SA. B, SAG.