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. 2012;7(10):e48443.
doi: 10.1371/journal.pone.0048443. Epub 2012 Oct 29.

Diuretics prime plant immunity in Arabidopsis thaliana

Yoshiteru Noutoshi  1 , Mika Ikeda, Ken Shirasu
Affiliations

Diuretics prime plant immunity in Arabidopsis thaliana

Yoshiteru Noutoshi et al. PLoS One. 2012.

Abstract

Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Molecular structures of the isolated diuretics as plant immune-priming compounds.
Figure 2
Figure 2. The effect of the diuretics on the pathogen-induced cell death of Arabidopsis suspension cells.
Arabidopsis suspension cultured cells were incubated with the compounds with or without the avirulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) avrRpm1, and the extent of cell death was quantitatively measured by the concentration of Evans blue dye. Each cell death rate is shown as a value relative to the mean of a mock treatment with the pathogen used for each experimental group. Sodium salicylate (SA) was used as a positive control. Error bars represent ±SE of four independent replicates. ** : P<0.01, * : P<0.05; Student's t-test with post-hoc Bonferroni's correction.
Figure 3
Figure 3. The increased disease resistance induced by the diuretics in Arabidopsis plants.
Arabidopsis seedlings were grown on rockwool for three weeks under short-day conditions, and their roots were drenched with water supplemented with 100 µM of the compounds for three days. As a positive control, 50 µM of sodium salicylate (SA) was used. Then, the avirulent Pst-avrRpm1 (A) and the virulent Pst (B) were inoculated into the leaves by syringe infiltration, and the bacterial numbers were counted at the indicated days (n = 4). **P<0.01; Student's t-test.
Figure 4
Figure 4. The expression of PR1 gene 24 hours after pathogen inoculation with or without the application of the diuretics.
The PR1 transcript level was determined by qRT-PCR. cDNA were prepared from seedlings 24 hours after inoculation of Pst-avrRpm1 with or without 100 µM of the diuretics. DMSO was used as mock treatment. The expression values were normalized to Actin2 as an internal standard. The bar represents the SE of three independent replicates. **P<0.01, *P<0.05; Student's t-test.
Figure 5
Figure 5. Effect of the diuretics on the growth of pathogenic bacteria.
Pst was cultured in liquid minimal medium supplemented with 200 µM of the indicated chemicals or 100 µg/mL hygromycin, and bacterial growth was monitored as the optical density of the bacteria at 600 nm at the indicated times after inoculation. The bar represents the SE of three independent replicates.
Figure 6
Figure 6. The expression of defense genes after the application of the compounds.
The mRNA transcript levels of PR1 and At3g57260 were determined by qRT-PCR using cDNA prepared from 10-day-old seedlings soaked in liquid media containing 100 µM of the chemicals for 24 or 48 hours. The expression values of the individual genes were normalized to Actin2 as an internal standard. These results are representative of three independent replicates. **P<0.01; Student's t-test.
Figure 7
Figure 7. Effect of the diuretics on the salicylic acid glucosyltransferase activity of UGT74F1.
The production levels of SA-β-D-glucoside (SAG) were measured by HPLC in the in vitro enzymatic reaction supplemented with 100 µM of the compounds using affinity-purified histidine-tagged recombinant UGT74F1 protein expressed in E. coli . Imprimatin A2 was used as a positive control. The data shown are relative to the DMSO control. The error bars represent the SE of independent triplicates. **P<0.01, *P<0.05; Student's t-test.
Figure 8
Figure 8. The effect of the diuretics on the germination and growth of Arabidopsis thaliana.
Arabidopsis seeds were dispensed into each well of a 96-well plate, and liquid media containing each chemical at the indicated concentrations was applied. Then, the plate was placed under long-day conditions at 22°C, and a photograph was taken after two weeks.
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MeSH terms

Grants and funding

This work was supported in part by KAKENHI (no. 22780036 to Y.N and 24228008 to K.S.) and the Special Coordination Fund for Promoting Sciences and Technology of MEXT to Y.N. This work was also partly supported by grants from The Sumitomo Foundation, The Kurata Memorial Hitachi Science and Technology Foundation and The Ryobi Teien Memory Foundation to Y.N. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.