Study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of Arabidopsis to microbial pathogens

Abstract

Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism. Two major fractions were obtained that appeared to have the same abundance in leaves from untreated plants versus leaves from plants challenge inoculated with the fungus Alternaria brassicicola. One of both major antimicrobial fractions was purified to homogeneity and identified by 1H nuclear magnetic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphinylbutyl isothiocyanate (ITC). This compound has previously been described as a product of myrosinase-mediated breakdown of glucoraphanin, the predominant glucosinolate in Arabidopsis leaves. 4-Methylsulphinylbutyl ITC was found to be inhibitory to a wide range of fungi and bacteria, producing 50% growth inhibition in vitro at concentrations of 28 microM for the most sensitive organism tested (Pseudomonas syringae). A previously identified glucosinolate biosynthesis mutant, gsm1-1, was found to be largely deficient in either of the two major antimicrobial compounds, including 4-methylsulphinylbutyl ITC. The resistance of gsm1-1 was compared with that of wild-type plants after challenge with the fungi A. brassicicola, Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, or Peronospora parasitica, or the bacteria Erwinia carotovora or P. syringae. Of the tested pathogens, only F. oxysporum was found to be significantly more aggressive on gsm1-1 than on wild-type plants. Taken together, our data suggest that glucosinolate-derived antimicrobial ITCs can play a role in the protection of Arabidopsis against particular pathogens.

Figure 1

HPLC profile obtained after loading Arabidopsis leaf extract from 4-g wild-type (Col-0) plants on a C18 reversed phase (RP)-HPLC column equilibrated in 0.1% (v/v) TFA. The column was eluted at a flow rate of 1 mL min⁻¹, 15 min with 0.1% (w/v) TFA, followed by a linear gradient of acetonitrile in 0.1% (v/v) TFA from 0% to 10% (v/v) acetonitrile in 30 min and from 10% to 45% (v/v) acetonitrile in the following 35 min. The eluate was monitored by online measurement of the A₂₃₉ (A₂₃₉; ——) and the acetonitrile gradient (−−−−−−) was monitored with an online conductivity sensor. Fractions (1.5 mL) were evaporated and dissolved in 60 μL of distilled water, of which 20 μL was assayed for antifungal activity (indicated as bars) against N. crassa as described in “Materials and Methods.” The indicated fractions A and B were used for further purification.

Figure 2

A, HPLC profile obtained after loading the combined antifungal fractions of Fraction B from Figure 1 on a C18 RP-HPLC column equilibrated in 0.1% (v/v) TFA. The column was eluted for 40 min with a linear gradient of acetonitrile in 0.1% TFA (v/v) from 0% to 40% (v/v) acetonitrile in 40 min at a flow rate of 1 mL min⁻¹. B, HPLC profile obtained after loading the indicated (arrow) peak fraction from Figure 2A, containing all antimicrobial activity, on a phenyl RP-HPLC column equilibrated in 0.1% (v/v) TFA. The column was eluted for 30 min with a linear gradient of acetonitrile in 0.1% (v/v) TFA from 0% to 30% (v/v) acetonitrile in 30 min at a flow rate of 1 mL min⁻¹. The indicated peak fraction (arrow) contained all antimicrobial activity. For both profiles, the eluate was monitored by online measurement of the A₂₃₉ (A₂₃₉; ——) and at the same time the acetonitrile gradient (−−−−−−) was monitored with an online conductivity sensor. Fractions (1.5 mL) were evaporated and dissolved in 60 μL distilled water, of which 20 μL was assayed for antifungal activity against N. crassa as described in “Materials and Methods.”

Figure 3

A, Light absorption spectrum of 4- methylsulphinylbutyl isothiocyanate. B, Mass spectrum of 4-methylsulphinylbutyl isothiocyanate obtained by gas chromatography (GC)/electron impact (EI) mass spectrometry (MS). C, Chemical structure of 4-methylsulphinylbutyl isothiocyanate.

Figure 4

HPLC profile obtained after loading Arabidopsis leaf extract from 4 g noninduced gsm1-1 leaf material on a C18 RP-HPLC column equilibrated in 0.1% (v/v) TFA. Elution of the column, fractionation, and determination of the antifungal activity as in Figure 1.

Figure 5

Comparative disease rating of glucosinolate-deficient gsm1-1 mutants and wild-type Col-0 plants inoculated with different pathogens. Throughout the different graphs, different letter labels indicate that the corresponding data are significantly different (P > 0.95) according to Tukey's studentized range test (Neter et al., 1996). A, Average diameter of necrotic lesions formed on leaves of 4-week-old Arabidopsis plants 6 d after drop inoculation with a spore suspension of A. brassicicola. Bars represent averages ± se of measurements from 40 lesions on five different plants. B, Average diameter of necrotic lesions formed on leaves of 4-week-old Arabidopsis plants 6 d after drop inoculation with a spore suspension of P. cucumerina. Bars represent averages ± se of measurements from 40 lesions on five different plants. C, Percentage from a total of eight fully expanded and inoculated leaves per plant showing chlorotic symptoms 8 d after inoculation with a spore suspension of F. oxysporum f. sp. matthiolae. Bars represent averages ± se from 20 4-week-old Arabidopsis plants. Analysis was done on leaves three through 10, with leaf numbering starting at one for the first true leaf and reflecting the order of appearance after germination. D, Percentage of inoculated leaves showing spreading necrosis symptoms 3 and 6 d after inoculation of Col-0 (●) and gsm1-1 (▵) plants with a spore suspension of B. cinerea. Data represent averages ± se of inoculations on all expanded leaves of 20 4-week-old Arabidopsis plants per genotype. E, Percentage of dead inoculated leaves 3 and 6 d after inoculation of Col-0 (●) and gsm1-1 (▵) plants with a bacterial suspension of E. carotovora. Data points represent averages ± se of inoculations on five expanded leaves of 20 4-week-old Arabidopsis plants. F, Growth of P. syringae pv tomato DC3000 in Col-0 (●) and gsm1-1 (▵) plants dip inoculated with a bacterial suspension of 10⁷ colony forming units (cfu) mL⁻¹. Data points represent averages ± se of log-transformed data from three experiments of the average number cfu per gram fresh leaf material.

Figure 6

Chlorosis symptoms of 4-week-old Arabidopsis Col-0 and gsm1-1 plants 8 d following inoculation on the leaves with F. oxysporum spores. Leaves are arranged from left to right in order of decreasing age. For each genotype, a series of eight leaves (third through 10th, in accordance with the scored leaves for the quantification of chlorosis symptoms as represented in Fig. 5C) from a representative plant is shown.

Figure 7

Quantification of F. oxysporum biomass in pooled (seventh through 10th) Col-0 and gsm1-1 infected leaves. Bars represent average relative fluorimetric values ± se of six samples, taken as described in “Materials and Methods,” 8 d following spray inoculation. Relative fluorimetric values were obtained by quantitative PCR, as described in “Materials and Methods.” Values are based on the quantification of a standard dilution series of DNA extracted from in vitro-grown F. oxysporum. Different letter labels indicate that the corresponding data are significantly different (P > 0.95) according to Tukey's studentized range test (Neter et al., 1996). This test was repeated with similar results.