Innate immunity in Arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes

Abstract

Lipopolysaccharides (LPS) are cell-surface components of Gram-negative bacteria and are microbe-/pathogen-associated molecular patterns in animal pathosystems. As for plants, the molecular mechanisms of signal transduction in response to LPS are not known. Here, we show that Arabidopsis thaliana reacts to LPS with a rapid burst of NO, a hallmark of innate immunity in animals. Fifteen LPS preparations (among them Burkholderia cepacia, Pseudomonas aeruginosa, and Erwinia carotovora) as well as lipoteichoic acid from Gram-positive Staphylococcus aureus were found to trigger NO production in suspension-cultured Arabidopsis cells as well as in leaves. NO was detected by confocal laser-scanning microscopy in conjunction with the fluorophore 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, by electron paramagnetic resonance, and by a NO synthase (NOS) assay. The source of NO was addressed by using T-DNA insertion lines. Interestingly, LPS did not activate the pathogen-inducible varP NOS, but AtNOS1, a distinct NOS previously associated with hormonal signaling in plants. A prominent feature of LPS treatment was activation of defense genes, which proved to be mediated by NO. Northern analyses and transcription profiling by using DNA microarrays revealed induction of defense-associated genes both locally and systemically. Finally, AtNOS1 mutants showed dramatic susceptibility to the pathogen Pseudomonas syringae pv. tomato DC3000. In sum, perception of LPS and induction of NOS contribute toward the activation of plant defense responses.

Fig. 1.

LPS induce a NO burst in Arabidopsis suspension cells. (A) Time course of the LPS-induced NO burst as detected by confocal laser-scanning microscopy. Arabidopsis cells were loaded with 5 μM DAF-FM DA and treated with buffer (Upper) or LPS (B. cepacia; 100 μg/ml) (Lower). Green fluorescence is indicative for NO. (Scale bars, 25 μm.) (B) LPS-induced increases of NO in Arabidopsis cells as detected by EPR. NO was detected by EPR by using the spin trap Fe₂ plus diethyldithiocarbamate. Shown are an extract obtained from untreated Arabidopsis cells, an extract from cells 10 min after LPS treatment, and a NO control (5 μM sodium nitroprusside in Hepes). The signals were recorded at identical EPR settings. (C) Time course of NO burst after LPS treatment. NO production was estimated by measuring fluorescence intensity with a microplate reader. The values (relative units) represent a mean of 25 independent experiments. (D) Effects of NOS and NR inhibitors on LPS-induced NO burst. Arabidopsis cells were treated with LPS and analyzed for NO by using 1 μM DAF-FM DA. In the case of inhibitor studies, cells were pretreated for 10 min with L-NNA or SoA before addition of LPS. Values represent a mean of five independent experiments.

Fig. 2.

Comparison of LPS-induced NO burst by diverse LPS preparations, Lipid A, and LTA. Cells were treated with the same concentration (100 μg/μl) of LPS, Lipid A, or LTA and/or 1 μM DAF-FM diacetate as described (Fig. 1 C and D). NO production was determined with a microplate reader (Fig. 1C). Values are expressed as NO production per minute and represent a mean of 10 independent experiments.

Fig. 3.

LPS elicited NO and NOS activity in Arabidopsis plants. (A) Fluorescence microscopy of LPS-induced increases in intracellular DAF-FM DA signals in epidermal cells from A. thaliana. The lower epidermis of Arabidopsis leaves was loaded with 1 μM DAF-FM DA in absence (Upper) or presence (B. cepacia; 100 μg/ml) (Lower) of LPS. The images were obtained 10 min after LPS treatment under bright field (c and g) and under fluorescence light (green light filter, 505–530 nm) (a, b, and d–f). Chlorophyll fluorescence was captured with a long-pass filter (585 nm) (b, d, and f). d shows an LPS-treated leaf coinfiltrated with the NO scavenger PTIO (1 mM). (Scale bars, 100 μm.) (B) LPS-induced NO in epidermal cells of Arabidopsis wild-type (WT), variantP-iNOS (varP), and AtNOS1 mutant plants. The NO burst was determined with a microplate reader during the first 60 min of treatment. The relative values represent a mean of four independent experiments. (C) NOS activity in wild-type Arabidopsis leaf extracts after LPS treatment. LPS-treated and control leaves were harvested at different time points, and an extract of leaf tissue was prepared. The NOS activity was determined with the NOS assay kit. Values represent a mean of four independent experiments.

Fig. 4.

DNA microarray analyses of transcriptional changes in A. thaliana plants (WT and AtNOS1) and suspension cells in response to LPS treatment. At the indicated time points after LPS treatment, mRNA from local and systemic leaf tissue or cells was hybridized to the cDNA array. A complete data set is presented in Table 1. Here, we present genes that respond to LPS in both test-systems (cells and leaves). White boxes, activation ≤1.5-fold; yellow, genes with >1.5- to <2.0-fold activation; light orange, 2.0- to <2.5-fold activation; orange, 2.5- to <3.0-fold activation; red, activation ≥3.0-fold. Greenish colors indicate repression. Gray numbers indicate weak signals <2-fold higher than surrounding background. The genes are arranged in alphabetical order.

Fig. 5.

Induction of local and systemic PR gene expression in Arabidopsis leaves by LPS. Arabidopsis leaves were treated with LPS (100 μg/ml) and collected at the times indicated for RNA preparation (4–48 h). Northern blots were probed with cDNAs for PR1, PR2, PR3, PR4, and PR5. Shown is the region between 1.8 and 1.0 kb. Ethidium bromide staining shows equal loading.

Fig. 6.

An AtNOS1 mutant shows enhanced disease susceptibility against Pst DC3000. Wild-type (WT) and AtNOS1 mutant plants were sprayed with Pst DC3000 bacteria or with water and photographed 2 (A) and 5 (B) days later, respectively. (Left) Symptoms after 2 and 5 days in a series of leaves. (Right) Bar graphs indicate the number of Pst DC3000 bacteria extracted from wild-type and AtNOS1 mutant plants 2 and 5 days after infection, respectively.