The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance

Abstract

Upon localized attack by necrotizing pathogens, plants gradually develop increased resistance against subsequent infections at the whole-plant level, a phenomenon known as systemic acquired resistance (SAR). To identify genes involved in the establishment of SAR, we pursued a strategy that combined gene expression information from microarray data with pathological characterization of selected Arabidopsis (Arabidopsis thaliana) T-DNA insertion lines. A gene that is up-regulated in Arabidopsis leaves inoculated with avirulent or virulent strains of the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) showed homology to flavin-dependent monooxygenases (FMO) and was designated as FMO1. An Arabidopsis knockout line of FMO1 proved to be fully impaired in the establishment of SAR triggered by avirulent (Psm avrRpm1) or virulent (Psm) bacteria. Loss of SAR in the fmo1 mutants was accompanied by the inability to initiate systemic accumulation of salicylic acid (SA) and systemic expression of diverse defense-related genes. In contrast, responses at the site of pathogen attack, including increases in the levels of the defense signals SA and jasmonic acid, camalexin accumulation, and expression of various defense genes, were induced in a similar manner in both fmo1 mutant and wild-type plants. Consistently, the fmo1 mutation did not significantly affect local disease resistance toward virulent or avirulent bacteria in naive plants. Induction of FMO1 expression at the site of pathogen inoculation is independent of SA signaling, but attenuated in the Arabidopsis eds1 and pad4 defense mutants. Importantly, FMO1 expression is also systemically induced upon localized P. syringae infection. This systemic up-regulation is missing in the SAR-defective SA pathway mutants sid2 and npr1, as well as in the defense mutant ndr1, indicating a close correlation between systemic FMO1 expression and SAR establishment. Our findings suggest that the presence of the FMO1 gene product in systemic tissue is critical for the development of SAR, possibly by synthesis of a metabolite required for the transduction or amplification of a signal during the early phases of SAR establishment in systemic leaves.

Figure 1.

A and B, Expression levels of FMO1 (At1g19250) in Arabidopsis leaves challenged with Pst according to microarray analyses. Means (±sd) of Affymetrix expression values originating from three independent replicates are given. The data were normalized according to the Affymetrix MAS 5.0 scaling protocol. A, Expression data from the NASC array NASCARRAYS-59: impact of type III effectors on plant defense responses. B, Expression data from TAIR (TAIR-ME00331: response to virulent, avirulent, type III secretion system-deficient and nonhost bacteria). C, RT-PCR analysis of FMO1 expression triggered by Psm (virulent strain) and Psm avrRpm1 (avirulent strain). Numbers indicate hpi. Control leaves (c) were infiltrated with 10 mm MgCl₂ for 24 h. 18S rRNA was amplified to standardize the transcript levels of each sample. D, Expression of FMO1 in leaves of wild-type and fmo1 mutant plants (T-DNA insertion line SALK_026163) 24 h after inoculation with Psm avrRpm1.

Figure 2.

A, Bacterial growth quantification of Psm in systemic leaves to assess SAR in wild-type and fmo1 mutant plants. Five-week-old Arabidopsis plants were pretreated with MgCl₂, Psm avrRpm1, or Psm (OD = 0.02 for each pathogen) in three primary leaves (1° treatment), and 2 d later, three systemic leaves located directly above the primary leaves were inoculated with Psm (OD = 0.002). Bacterial growth in systemic leaves was assessed 3 d (3 dpi) after infection of systemic leaves. Bars represent mean values (±sd) of colony-forming units per square centimeter from seven parallel samples each consisting of three leaf discs. Asterisks denote pathogen treatments with statistically significant differences to the respective MgCl₂ control (P < 0.001; Student's t test). Light bars, Wild-type plants; dark bars, fmo1 plants. B and C, Quantification of bacterial growth to assess local resistance. B, Growth of Psm avrRpm1 in leaves 3 d after inoculation with a bacterial suspension of OD = 0.005. C, Growth of Psm in leaves 3 d after inoculation (OD = 0.002). In both B and C, no statistical differences between the wild type and fmo1 existed (P > 0.05; Student's t test). In addition, to ensure the uniformity of the experiments, initial bacterial numbers (1 hpi) were quantified. No significant differences in bacterial numbers were detected at 1 hpi for comparable treatments in A, B, and C (data not shown).

Figure 3.

Systemic defense responses in wild-type and fmo1 plants. Primary leaves of 5-week-old plants were treated as described in Figure 2A and untreated systemic leaves were harvested 2 d later for analysis. A, Systemic accumulation of SA. Bars represent mean values (±sd) of three independent samples. Each sample consisted of six leaves from two different plants. Asterisks denote pathogen treatments with statistically significant differences to the respective MgCl₂ control (*, P < 0.02; **, P < 0.005; Student's t test). White bars, MgCl₂ treatment; gray bars, Psm avrRpm1 inoculation; black bars, Psm inoculation. B, Systemic expression of defense-related genes assessed by northern-blot analysis (c, MgCl₂ treatment; a, Psm avrRpm1 inoculation; v, Psm inoculation).

Figure 4.

Local defense responses in wild-type and fmo1 plants. A to C, Accumulation of signaling and antimicrobial compounds in leaves challenged with Psm avrRpm1 (OD = 0.005). Control samples were treated with 10 mm MgCl₂. All samples were collected 10 h post treatment. A, SA levels. B, JA content. C, Accumulation of the phytoalexin camalexin. Mean values (±sd) of three independent samples are given. No statistical differences between equally treated wild-type and fmo1 plants existed for each metabolite (P > 0.05; Student's t test). D, Quantification of microscopic HR lesions in leaves inoculated with Psm avrRpm1 that were stained with trypan blue 24 hpi. Bars represent mean values (±sd) of dead cells in infiltrated areas from at least seven independent leaf samples. Light bars, Areas infiltrated with 10 mm MgCl₂; dark bars, Psm avrRpm1-infiltrated areas.

Figure 5.

Local defense responses in wild-type and fmo1 plants. Expression of defense-related genes in leaves challenged with Psm avrRpm1 (OD = 0.005), as assessed by northern-blot analysis. Numbers indicate hpi. Control leaves (c) were treated with 10 mm MgCl₂ (4 h).

Figure 6.

A, Expression of FMO1 at the site of pathogen inoculation in wild-type plants and Arabidopsis defense mutants (24 hpi) as assessed by RT-PCR analysis (c, MgCl₂ treatment; a, Psm avrRpm1 inoculation; v, Psm inoculation; OD = 0.005 for each pathogen). B, Levels of FMO1 transcripts in untreated leaves of wild-type and cpr5 mutant plants.

Figure 7.

Correlation of systemic FMO1 expression and SAR establishment. A, Systemic expression of FMO1 in wild-type plants and Arabidopsis defense mutants in response to Psm avrRpm1 as assessed by RT-PCR analysis (c, MgCl₂ treatment; a, Psm avrRpm1 inoculation; OD = 0.02). For further details, see legend to Fig. 3. B, Growth quantification of Psm in systemic leaves (3 dpi) to assess SAR induced by Psm avrRpm1 in wild-type and Arabidopsis defense mutants. For further details, see legend to Fig. 2A. Asterisks denote lines with statistically significant differences between plants pretreated with MgCl₂ and Psm avrRpm1 (*, P < 0.01; **, P < 0.001; Student's t test).