Vitamin B1 functions as an activator of plant disease resistance

Abstract

Vitamin B(1) (thiamine) is an essential nutrient for humans. Vitamin B(1) deficiency causes beriberi, which disturbs the central nervous and circulatory systems. In countries in which rice (Oryza sativa) is a major food, thiamine deficiency is prevalent because polishing of rice removes most of the thiamine in the grain. We demonstrate here that thiamine, in addition to its nutritional value, induces systemic acquired resistance (SAR) in plants. Thiamine-treated rice, Arabidopsis (Arabidopsis thaliana), and vegetable crop plants showed resistance to fungal, bacterial, and viral infections. Thiamine treatment induces the transient expression of pathogenesis-related (PR) genes in rice and other plants. In addition, thiamine treatment potentiates stronger and more rapid PR gene expression and the up-regulation of protein kinase C activity. The effects of thiamine on disease resistance and defense-related gene expression mobilize systemically throughout the plant and last for more than 15 d after treatment. Treatment of Arabidopsis ecotype Columbia-0 plants with thiamine resulted in the activation of PR-1 but not PDF1.2. Furthermore, thiamine prevented bacterial infection in Arabidopsis mutants insensitive to jasmonic acid or ethylene but not in mutants impaired in the SAR transduction pathway. These results clearly demonstrate that thiamine induces SAR in plants through the salicylic acid and Ca(2+)-related signaling pathways. The findings provide a novel paradigm for developing alternative strategies for the control of plant diseases.

Figure 1.

Effects of thiamine application on disease progress in rice, tobacco, cucumber, and Arabidopsis. Plants were inoculated with each pathogen at 4 h after spraying of mock (control, 250 μg mL⁻¹ Tween 80) or thiamine (thiamine, 50 mm in 250 μg mL⁻¹ Tween 80) solutions. A, The necrotic lesions normally caused on the rice cultivar Hwacheong by the rice blast disease pathogen M. grisea strain KJ201 (5 × 10⁵ conidia mL⁻¹) are suppressed in thiamine-treated leaves. The leaves were photographed at 7 d post inoculation. The graph shows the protection against blast disease provided by the pretreatment with thiamine. The disease severities were evaluated daily as described in “Materials and Methods.” Each point represents the mean ± se of 10 plants. B, The yellow lesions normally caused on the rice cultivar Nakdong by the rice bacterial leaf blight pathogen X. oryzae pv oryzae strain KXO21 (1 × 10⁸ CFU mL⁻¹) are suppressed in thiamine-treated rice plants. The leaves were photographed at 10 d post inoculation. The graph shows the reduced lesion lengths observed after pretreatment with thiamine. Each curve represents the mean ± se of 10 plants. C, Thiamine acts as an antiviral compound against PMMoV infection in the genetically susceptible tobacco cultivar Samsun NN. The second true leaf of mock- or thiamine-treated plants was inoculated with PMMoV and photographed at 10 d post inoculation. Three inoculated leaves were harvested at 0, 1, 2, 3, and 4 d post inoculation for RNA extraction. Northern-blot hybridization analyses were conducted using an RT-PCR product of PMMoV labeled with [³²P]dCTP as a probe. Equal sample loading was confirmed by ethidium-bromide staining of the rRNA in the gel. D, The necrotic lesions normally caused on the cucumber cultivar Sunmi-Baekdadaki by the cucumber anthracnose pathogen C. lagenarium are suppressed in thiamine-treated cucumber plants. The graph shows the levels of disease protection observed in thiamine-treated plants and untreated controls. The percentage of the symptomatic area was evaluated at 7 d after inoculation and photographed as described in “Materials and Methods.” Each bar represents the mean ± se of five plants. E, The necrotic lesions normally caused on Arabidopsis ecotype Col-0 by the Arabidopsis pathogen Pst DC 3000 are suppressed in thiamine-treated plants. Representative samples were collected from 10 plants at 1, 2, 3, 4, and 5 d after spraying with a bacterial solution of 1 × 10⁸ CFU mL⁻¹.

Figure 2.

Defense-related gene expression induced by thiamine treatment and pathogen inoculation. Pathogen inoculation and thiamine treatment were performed as described in Figure 1. dpi, Days post inoculation. A, Rice (cv Hwacheong) defense-related gene expression induced by spraying with a thiamine solution and/or infection by M. grisea strain KJ201. Total RNA was extracted from the leaves of five rice plants at 0, 1, 2, 3, and 4 d after inoculation with M. grisea. B, Rice (cv Nakdong) defense-related gene expression induced by spraying with a thiamine solution and/or infection with X. oryzae pv oryzae strain KXO21 (Xoo). Total RNA was extracted from the leaves of five rice plants at 0, 1, 2, 3, and 4 d post inoculation with Xoo. C, Defense-related gene expression induced in tobacco by spraying with a thiamine solution and/or infection with PMMoV. Total RNA was extracted from the second true leaves of five tobacco plants at 0, 1, 2, 3, and 4 d following inoculation with PMMoV. D, Expression of the cucumber acidic peroxidase gene (POX) induced by spraying with a thiamine solution and/or infection with C. lagenarium. Total RNA was extracted from the second and third true leaves of five cucumber plants at 0, 1, 2, 3, and 4 d following inoculation with C. lagenarium. Total RNA was extracted, separated by denaturing gel electrophoresis, and transferred to nylon membrane. The blots were hybridized with probes labeled with [³²P]dCTP. Equal sample loading was confirmed by ethidium-bromide staining of the rRNA in the gel.

Figure 3.

Specificity of the activation of plant defenses by thiamine. Rice plants were sprayed with solutions of TMP (1 mm TMP with 250 μg mL⁻¹ Tween 80) or TPP (1 mm TPP with 250 μg mL⁻¹ Tween 80). The rice cultivars Hwacheong and Nakdong were inoculated with the M. grisea strain KJ201 or the X. oryzae pv oryzae strain KXO21 at 4 h after TMP and TPP treatments, respectively. A, Effects of TMP and TPP on the progress of rice blast disease and bacterial leaf blight disease. The progress of rice blast disease and lesion lengths of plants with bacterial leaf blight disease were evaluated at 7 d after inoculation and photographed as described in “Materials and Methods.” Each bar represents the mean ± se of 10 plants. B, Rice PR-1 gene expression induced by infection with M. grisea or X. oryzae pv oryzae (Xoo, mock) in the presence or absence of 1 mm TMP or 1 mm TPP. Total RNA was extracted from rice leaves harvested from five rice plants, separated using denaturing gel electrophoresis, and transferred to nylon membrane. The blots were hybridized with a rice PR-1 probe labeled with [³²P]dCTP. Equal sample loading was confirmed by ethidium-bromide staining of the rRNA in the gel or hybridization with a radioactive 18S rRNA probe after removal of the PR-1 probe.

Figure 4.

Thiamine suppresses rice blast disease for up to 15 d following treatment through the induction of resistance responses. The rice cultivar Hwacheong was inoculated with the rice blast pathogen M. grisea strain KJ201 at 3, 7, and 15 d after thiamine treatment. The disease severity was evaluated daily as described in “Materials and Methods.” A, Rice blast disease progress in mock-treated and thiamine-treated rice leaves following M. grisea infection at 3, 7, and 15 d after treatment. Each bar represents the mean value from 10 plants. White bars, Mock-treated rice leaves; black bars, thiamine-treated rice leaves. B, Expression patterns of defense-related genes in thiamine-treated rice leaves inoculated with M. grisea at 7 and 15 d after thiamine treatment. Total RNA was extracted from the leaves of three plants at 0, 1, 2, 3, and 4 d after fungal inoculation, separated by denaturing gel electrophoresis, and transferred to nylon membrane. The blots were hybridized with a rice PR-1 probe labeled with [³²P]dCTP. Equal sample loading was confirmed by ethidium-bromide staining of the rRNA in the gel.

Figure 5.

Analysis of PR-1 gene expression and quantification of resistance to Pst DC 3000 infection following thiamine treatment of Arabidopsis Col-0, nahG, npr1, etr1, and jar1 plants. A, Effect of thiamine on the accumulation of PR-1 transcripts in Arabidopsis. Total RNA was extracted from the leaves of five plants at 24 h after thiamine treatment, separated by denaturing gel electrophoresis, and transferred to nylon membrane. The blots were hybridized with an Arabidopsis PR-1 probe labeled with [³²P]dCTP. Equal sample loading was confirmed by ethidium-bromide staining of the rRNA in the gel. B, Numbers of Pst DC 3000 in leaves of the Arabidopsis ecotype Col-0 and signaling mutants of the same ecotype treated with thiamine 4 h prior to bacterial inoculation. Samples were collected from five wild-type and five mutant plants at 3 d after inoculation, and all experiments were conducted three times independently, with three replicates. White bars, Mock-treated Arabidopsis leaves; black bars, thiamine-treated Arabidopsis leaves. The data represent the mean ± se of three pools of five plants. The experiments were repeated at least five times, with similar results each time.

Figure 6.

Accumulation of the PR-1 transcript in Arabidopsis ecotype Col-0 and induction of PKC activity in rice triggered by thiamine treatment and/or pathogen inoculation. A, Systemic expression of PR-1 induced by thiamine treatment of the Arabidopsis ecotype Col-0. Thiamine was sprayed only on the rosette (T), and the upper cauline leaves were left untreated (U). Total RNA was extracted from rosette and cauline leaves of five plants harvested at 24 h after thiamine treatment. Total RNA was also extracted from thiamine-untreated leaves (C). The blots were hybridized with an Arabidopsis PR-1 gene probe labeled with [³²P]dCTP. Equal sample loading was confirmed by ethidium-bromide staining of the rRNA in the gel. B, Thiamine treatment increases PKC activity in rice following infection with M. grisea. Plants of the rice cultivar Hwacheong that had been treated with thiamine or mock treated were inoculated with M. grisea at 4 h after thiamine treatment. Abbreviations: C, Treated with 250 μg mL⁻¹ Tween 80 only; T, treated with 50 mm thiamine; I, infected with M. grisea; T + I, infected with M. grisea after thiamine treatment. Total protein was extracted from the leaves of five plants harvested at 24 h after the thiamine treatment. C, Infiltration of Arabidopsis plants with LaCl₃, a calcium channel blocker, resulted in the suppression of PR-1 gene induction by thiamine. Abbreviations: C, Infiltrated with distilled water at 4 h after spraying with 250 μg mL⁻¹ Tween 80; T, infiltrated with distilled water at 4 h after spraying with 50 mm thiamine; T + L, leaves infiltrated with 1 mm LaCl₃ at 4 h after spraying with 50 mm thiamine. Total RNA was extracted from the leaves of five plants at 24 h after thiamine treatment, separated using denaturing gel electrophoresis, and transferred to nylon membrane. The blots were hybridized with an Arabidopsis PR-1 probe labeled with [³²P]dCTP. Equal sample loading was confirmed by ethidium-bromide staining of the rRNA in the gel.