Potentiation of pathogen-specific defense mechanisms in Arabidopsis by beta -aminobutyric acid

Abstract

The nonprotein amino acids gamma-aminobutyric acid (GABA) and beta-aminobutyric acid (BABA) have known biological effects in animals and plants. Their mode of action has been the object of thorough research in animals but remains unclear in plants. Our objective was to study the mode of action of BABA in the protection of Arabidopis plants against virulent pathogens. BABA protected Arabidopsis against the oomycete pathogen Peronospora parasitica through activation of natural defense mechanisms of the plant such as callose deposition, the hypersensitive response, and the formation of trailing necroses. BABA was still fully protective against P. parasitica in transgenic plants or mutants impaired in the salicylic acid, jasmonic acid, and ethylene signaling pathways. Treatment with BABA did not induce the accumulation of mRNA of the systemic acquired resistance (SAR)-associated PR-1 and the ethylene- and jasmonic acid-dependent PDF1.2 genes. However, BABA potentiated the accumulation of PR-1 mRNA after attack by virulent pathogenic bacteria. As a result, BABA-treated Arabidopsis plants were less diseased compared with the untreated control. In the case of bacteria, BABA protected mutants insensitive to jasmonic acid and ethylene but was not active in plants impaired in the SAR transduction pathway. Thus, BABA protects Arabidopsis against different virulent pathogens by potentiating pathogen-specific plant resistance mechanisms. In addition, we provide evidence that BABA-mediated papilla formation after P. parasitica infection is independent of the SAR signaling pathway.

Figure 1

BABA-induced resistance in Arabidopsis against P. parasitica. (A) Microscopic aspects of the protective effect of BABA. (I) Growing hyphae in untreated control plant. (Bar = 50 μm.) (II) Callose (yellow) deposition (p, papilla) below the appressoria (ap) at the end of the germ tube (gt) on leaf treated with 12 mg/liter BABA. c, conidium. (Bar = 20 μm.) (III) Phenocopy–HR reaction in plants treated with 8 mg/liter BABA. Callose deposition (arrowheads) around the appressoria and cells undergoing necrosis appear in yellow. (Bar = 20 μm.) (IV) Trailing necrosis (arrowheads) along a growing hypha (h) in a plant treated with 4 mg/liter BABA. (Bar = 50 μm.) Plants were treated with BABA 1 day before inoculation and stained 3 days later with aniline blue for callose observation (Wasserblau Standard Fluka) (30) and Calcofluor White M2 R. S. New (Cyanamid) (44) (II and III) or with lactophenol-trypan blue (I and IV) (45) for fungal structure coloration. Picture IV was taken 6 days after inoculation. (B) Effect of BABA in Arabidopsis lines altered in their response to P. parasitica. Wild-type (Col-0) control, NahG, npr1, jar1, and etr1 plants were treated with water or 12 mg/liter BABA and inoculated with the virulent P. parasitica isolate NOCO. Pictures show leaves stained with lactophenol-trypan blue (45) 7 days after inoculation. Fungal structures and damaged cells are stained in blue. Genotypes and treatments are indicated in the middle and top of the figure, respectively. A representative example for each genotype is shown.

Figure 2

Time course of the expression of PR-1 mRNA in Arabidopsis. (A) Effect of chemical treatments. Total RNA was extracted at various times after being soil drenched with water, 16 mg/liter BABA, or 300 μM benzothiadiazole. hpt, hours posttreatment. (B) Conditioning effect of BABA. Plants were soil drenched with water or 16 mg/liter BABA 1 day before infiltration with bacteria (time 0). Each time point represents nine infected leaves harvested from three different plants. Total RNA was prepared and analyzed by RNA gel blot analysis. Ethidium bromide staining of the RNA gel (rRNA) was used to show equal loading. hpi, hours postinoculation.

Figure 3

Protection effect of BABA in Arabidopsis infected with Pst DC 3000. (A) Bacterial growth. Wild-type Col-0, NahG, npr1, jar1, and etr1 leaves were analyzed for bacterial density at different time points after infiltration. Data represent the mean ± SE of three pools stemming from eight replicate samples. Experiments were repeated four times with similar results. (B) Symptoms. Pictures show disease symptoms 3 days after infiltration in wild-type Col-0, NahG, npr1, jar1, and etr1 plants. Treatments are indicated at the top of the figure. Representative examples are shown.