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

Crude aqueous extracts from Arabidopsis leaves were subjected to chromatogr aphic separations, after which the different fractions were monitored for a ntimicrobial activity using the fungus Neurospora crassa as a test organism . Two major fractions were obtained that appeared to have the same abundanc e in leaves from untreated plants versus leaves from plants challenge inocu lated with the fungus Alternaria brassicicola. One of both major antimicrob ial fractions was purified to homogeneity and identified by H-1 nuclear mag netic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphin ylbutyl isothiocyanate (ITC). This compound has previously been described a s a product of myrosinase-mediated breakdown of glucoraphanin, the predomin ant glucosinolate in Arabidopsis leaves. 4-Methylsulphinylbutyl ITC was fou nd to be inhibitory to a wide range of fungi and bacteria, producing 50% gr owth inhibition in vitro at concentrations of 28 muM for the most sensitive organism tested (Pseudamonas syringae). A previously identified glucosinol ate biosynthesis mutant, gsm1-1, was found to be largely deficient in eithe r of the two major antimicrobial compounds, including 4-methylsulphinylbuty l 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 b acteria 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-deriv ed antimicrobial ITCs can play a role in the protection of Arabidopsis agai nst particular pathogens.